Methods of Detecting Conjugation Site-Specific and Hidden Epitope/Antigen

ABSTRACT

This invention discloses “Artificially Cleaved Epitope (ACE)” methods, antibodies, reagents, immunoassays, and kits for designing and detecting hidden epitopes/antigens. The ACE methods can detect epitopes that are either absent or poorly accessible naturally to antibodies, and thus must be specifically and artificially created (free terminals) and/or exposed in samples and sample preparations for antibody detection. The ACE structures include, but are not limited to, macromolecule-to-macromolecule conjugation sites, and any types of linear hidden epitopes. The ACE methods comprise ACE antigen design and ACE antigen detection. The ACE methods, antibodies, reagents, immunoassays, and kits are useful in research and discovery, diagnostic, and therapeutic applications. In another aspect, the ACE methods can artificially and specifically expose hidden antigens while reducing the antibody non-specific bindings in all antibody-based applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/312,285, filed Dec. 6, 2011, which claims priority to Ser. No.61/420,354 filed on Dec. 7, 2010, both incorporated herein by referencein their entirety.

TECHNICAL FIELD

The present description relates to methods, antibodies, reagents,immunoassays and kits for designing and detecting hidden antigens,including, but not limited to macromolecule-to-macromolecule conjugationsites or any types of hidden antigens, either in situ or ex situ insample preparations, collectively defined as the ACE methods, and theiruse thereof.

BACKGROUND OF THE INVENTION

It is estimated that about 50,000 -100,000 antibodies are currentlyavailable worldwide representing about 5,000 -10,000 differentgenes/proteins. This number is significantly below 400,000 proteins inthe proteome, and millions of other “antibodiable” antigens.Furthermore, most antibodies don't work as the users intend, and about75% of antibodies do not work in all antibody-based applications(http://www.sdix.com/Products/Custom-Antibody-Services/Engagement/Antibodies.aspx?Q=2).A key reason of much fewer antibodies than “antibodiable” antigens isthat many antigens are hidden in samples or sample preparations and thuspoorly accessible to antibodies. This invention discloses a method ofdesigning and detecting hidden antigens.

Post-translational conjugation of a protein by another protein,polysaccharide, lipid and nucleic acid, or any combination of the aboveplays a key role virtually in every aspect of cellular functions. Aconjugated molecule is either a monomeric single-molecule or a polymericmacromolecule with either a linear or a branched structure.Macromolecules include, but are not limited to, proteins orpolypeptides, polysaccharides, adenosine diphosphate (ADP)-ribosyls,fatty acids, polynucleotides, glycosylphosphatidylinositol (GPI)anchors, ubiquitin, small ubiquitin-like modifier (SUMO), neuralprecursor cell expressed, developmentally down-regulated 8 (NEDD8),interferon-stimulated gene 15 kDa (ISG15), and other ubiquitin-likemolecules (UBLs).

Many types of human diseases display abnormal molecular conjugation. Forexample, abnormal glycosylation occurs in many types of cancers (Mehtaand Block, 2008). Ubiquitin-containing conjugates are present virtuallyin all types of neurodegenerative diseases (Dohm et al., 2008).Telomeric aggregates accumulate in tumor cells (Mai and Garini, 2006).Advanced glycation adducts are found in samples obtained from patientswith heart disease and/or diabetes (Thornalley, 2002; Meerwaldt et al.,2008). Disease-specific macromolecule-to-macromolecule conjugates arepresent in body fluids such as blood serum or cerebrospinal fluid (CSF),but few reliable methods are currently available to detect them.However, most, if not all, macromolecule-to-macromolecule conjugationsites are hidden antigens (see FIG. 4 below). Therefore, antibodies tomacromolecule-to-macromolecule conjugation sites are difficult to makeand are not currently available for assays ofmacromolecule-to-macromolecule conjugation sites in all antibody-basedapplications.

Methods of making antibodies against post-translational modifiedproteins in the form of a small monomeric molecule, includingphosphorylation, acetylation, methylation, and nitrolization, are wellestablished. In comparison to monomeric modification site-specificantibodies, there is no effective method currently available for makingpolymeric macromolecular conjugation site-specific antibodies.Macromolecular conjugation can be defined as covalent conjugationbetween two polymeric biomolecules, including, but not limited to,protein glycosylation, lipidation, ADP-ribosylation, ubiquitination,sumoylation, NEDDylation, ISGylation, GPI-anchor,transglutaminase-mediated cross-links, and the like.

In the post-genomic era, our knowledge of macromolecule-to-macromoleculeconjugation and its relation to diseases has grown exponentially. Thisprovides an opportunity to develop novel methods for detectingmacromolecule-to-macromolecule conjugation in a conjugationsite-specific manner. For that reason, investigators have devotedextensive efforts to generation of macromolecule-to-macromoleculeconjugation site-specific antibodies by conventional antigen design,antibody-making, and antigen detection methods. However, these effortshave been so far proven futile (Matsumoto et al., 2008). As a result,there are few conjugation site-specific antibodies currently available.Therefore, new methods for detecting hidden antigen/epitopes, includingbut not limited to macromolecular conjugation-sites and linear hiddenantigens, are desperately needed, and can provide useful tools for allantibody-and antigen-based applications.

An example is making ubiquitin-to-protein conjugation site-specificantibodies. Protein ubiquitination involves virtually all proteindegradation as well as other biological processes. There are a fewprevious reports of generation of anti-polyubiquitin antibodies. Pirim(1998) reported an anti-polyubiquitin antibody. However, this antibodydoes not recognize isopeptide bond-branched ubiquitin-to-ubiquitinconjugation, which are dominant forms of cellular ubiquitin conjugates.Rather this antibody recognizes head-to-tail (c- to n-terminalconjugation) poly-ubiquitins, which represent a very tiny/small fractionof polyubiquitin transiently formed during synthesis of free ubiquitin.

Fujimuro et al. (2005) reported anti-polyubiquitin monoclonal antibodiesnamed as FK1 and FK2. Both FK1 and FK2 antibodies recognize thepolyubiquitin chain. However, there are two fundamental differencesbetween making FK1 and FK2 antibodies and the methods described in theclaims of the present invention: (i) FK1 and FK2 were made by usingregular polyubiquitin antigens; and (ii) FK1 and FK2 cannot recognizespecific conjugation sites of ubiquitinated proteins (Fujimuro et al.,2005). Therefore, FK1 and FK2 are not conjugation site-specificantibodies, rather than general polyubiquitin antibodies.

Similarly, there are reports of using antibodies to theglycine-glycine-to-(lysine) structure for profiling of ubiquitinatedproteins and identification of the peptides with liquidchromatography-tandem mass spectrometry (LC-MS/MS) (Peng et al., 2003;Denis et al., 2007; Xu et al., 2010). The glycine-glycine-to-(lysine)structure was prepared by reacting lysine-rich histone III-S proteinwith t-butyloxycarbonyl-Gly-Gly-N-hydroxysuccinimide (Boc-Gly-Gly-NHS)(Xu et al., 2010). However, the glycine-glycine-to-(lysine) antibodiesrecognize only the glycine-glycine-to-(lysine), rather than the lysinesurrounding sequence of a specific ubiquitinated protein, and thus theyare also not conjugation site-specific antibodies, and cannot be used todetect individual ubiquitin-to-protein conjugation sites. In addition,the glycine-glycine-to-(lysine) antibodies cannot be used in regularantibody-based applications, rather they were developed for thepre-LC-MS/MS profiling applications (Peng et al., 2003; Denis et al.,2007; Xu et al., 2010). In comparison, the inventive methods are fordesigning and detecting the specific conjugation sites of bothconjugation moieties. Therefore, conjugation site-specific antibodiesdeveloped by the inventive ACE methods, can recognize specifically boththe branched glycine-glycine and the conjugation site lysine surroundingsequence as well as can be used for detecting hidden antigens in allantibody-based applications (see FIGS. 1-13).

Matsumoto et al. (2008) generated two linkage-specific antibodies thatrecognize polyubiquitin chains through lysine 63 (K63) or 48 (K48)linkage (US patent 20070218069A). However, there are several fundamentaldifferences between the method of making these two linkage-specificantibodies and the methods of the present invention. The “antibodies”made by Matsumoto et al. (2008) were not generated by conventionalanimal immunization methods, rather by a phage display approach ofrandom screening of the ubiquitin conjugation site binders. This phagedisplay approach has advantage to be able to select binding partnersfrom millions of other irrelevant proteins, but these binding partnersare “antibody-like” fusion proteins. Also, the phage display approachusually has technical challenges associated with it. For instance, it isacknowledged that the affinity and specificity of binding partnersgenerated by phage display are often suboptimal, relative toconventional antibody, and the loss of the original heavy-andlight-chain pairings is also a challenge. Perhaps for these reasons,phage display has not been widely used to make “antibodies” (Ward,2002). In comparison, the present invention uses the ArtificiallyCleaved Epitope (ACE, see below) strategy for designing and detectingmacromolecular conjugation site-specific and linear hidden antigens,which are proven to be more effective and reliable (see FIGS. 1-14below).

There are several patented methods for making peptide antibodies. PatentWO 02/25287 describes methods for analysis of proteins by producing amixture of peptides and contacting the mixture of peptides to filteringagents or antibodies in order to decrease the complexity of a mixtureprior to the application of an analytical technique such as massspectrometry. U.S. Pat. No. 7,460,960 described methods by the use ofcapture agents or antibodies that interact with the Proteome EpitopeTags (PETs) in a sample. However, these methods cannot be used to designand detect hidden antigens, and they are also principally and profoundlydifferent with the methods of the present invention.

Currently, there are several cleavage site-specific antibodiescommercially available. U.S. Pat. No. 7,803,553 by Kojima et al.described an antibody for detecting an active form of TGF-β1 naturallycleaved in vivo. U.S. Pat. No. 6,762,045 by Krebs et al. described anantibody to naturally cleaved caspase-3. All currently availablecleavage-specific antibodies were developed to detect the naturallyoccurring cleavage sites in vivo, and cannot be used to detect hiddenantigens such as macromolecule-to-macromolecule conjugation sites. Incontrast, the present inventive methods are to design and detect“Artificially Cleaved Epitopes (ACEs, see below)” of hidden antigensthat are not naturally present or exposed. The inventive ACE methods donot include those for detecting naturally cleaved epitopes in samples.

Macromolecules other than polypeptides can also be used to generateantibodies successfully, including, but not limited to, antibodies tolipids, nucleic acids, and saccharide. For example, a mouse monoclonalantibody (e.g., CTD110.6) recognizing the single O-linkedN-acetylglucosamine is commercially available. A mouse antibody (e.g.,clone 26-5) to a lipid structure is also reported (Young et al., 1987).However, polysaccharide-to-protein and lipid-to-protein conjugationsite-specific antibodies are not currently available, most probablybecause they are hidden antigen and no reliable methods can successfullymake them.

SUMMARY OF THE INVENTION

The present invention discloses the “Artificially Cleaved Epitope” orACE methods, antibodies, reagents, immunoassays and kits for designingand detecting hidden antigens, collectively defined as the ACE methods,and their use thereof.

The ACE methods encompass all or part of these steps: (a) Make an ACEantigen by designing, synthesizing or isolating the ACE structure; (b)Make ACE antibody with the ACE antigen by any antibody-making,antibody-like molecule-making methods, and the like; (c) The ACEstructure in sample preparations is not naturally or poorly accessibleby antibody, and thus must be exposed artificially and precisely eitherin situ or ex situ by the designated hydrolytic enzyme or chemical agenttreatment in a sequence-dependent and residue chemical bond-specificmanner; and (d) Detect ACE in situ or ex situ by any antibody-basedmethod in any types of sample preparations. This invention also includesutilities and applications of the ACE methods, antibodies, reagents,immunoassays and kits.

In one embodiment, this invention provides unique ACE antigen designmethods, wherein the ACE structure must possess one or combinations ofthese characters: (a) must be antigenic, (b) must be a complete ortruncated form of an artificially chemical bond-specific hydrolyticenzyme-or agent-cleaved segment (i.e., ACE); (c) either a branchedconjugation site derived from both conjugation moieties, or a linearhidden antigen segment that is folded/buried inside its parent orsurrounding macromolecule/structures; (d) must be artificially andspecifically created and/or exposed in a sequence-dependent and residuechemical bond-specific manner in samples or sample preparations (e.g.,in Western blot membranes, tissues or cell lysates, tissue sections,isolated or culture cells, isolated fractions, any ACE-containingsurfaces/matrices/materials, and the likes); and (e) the hiddenconjugation sites or linear hidden epitopes may be amino acids/peptides,sugar monomers/polymers, lipids/lipid linkers (e.g., ethanolamine),nucleic acids, ADP-ribose, or their combinations.

As mentioned, most macromolecule-to-macromolecule conjugation sites arehidden antigens. A general hidden macromolecule-to-macromoleculeconjugation site-specific ACE structure is:Ln---L2-L1(-S1-S2---Sm)-L1′-L2′---Lm′, wherein the ACE structure is abranched segment of an intact macromolecule-to-macromolecule conjugatecomposed of L and S oligomeric residue chains (e.g., peptides,saccharides, lipids, nucleic acids, ADP-ribosyls, or their conjugates);wherein S1 is covalently conjugated to L1 sidechain; wherein Ln, Lm′, orSm are continuously counted from L1, L1′ or S1 residues; wherein Ln,Lm′, or Sm are not free ends naturally, and have covalent chemical bondswith residues outside of the hidden ACE structure; and wherein saidcovalent chemical bonds must be artificially and specifically cleaved byspecifically designed and chemical bond-specific hydrolysis in samplepreparations to create and/or expose said hidden ACE structure forantibody detection.

The designs of hidden conjugation site-specific ACE structures ofSUMO-to-protein, ubiquitin-to-protein, saccharide-to-protein, fattyacid-to-protein, GPI-to-protein, transglutaminase-mediated crosslink,and the like, are shown in FIGS. 1-8. By using the inventive ACEmethods, we have successfully made several ACE antibodies that arehighly specific to individual hidden macromolecule-to-macromoleculeconjugation sites in biological samples including alpha-fetoprotein(AFP) core-fucosylation, ubiquitin-to-histon 2A K120, Na⁺/K⁺ ATPase, andubiquitin-to-ubiquitin K48 conjugation site-specific antibodies, asshown in FIGS. 3, and 9-13.

In addition to macromolecular conjugation site, the inventive ACEmethods can also be used to design and detect linear hidden antigenseither in situ or ex situ in a sample preparation. An linear hidden ACEstructure is “L1 -L2 ------Ln” which is a hidden segment of an intactpolymeric macromolecule (e.g., polypeptide, polysaccharide, lipid,nucleic acids, ADP-ribosyls, or their conjugates), wherein L1 and Ln arenot free ends or terminals, and have covalent chemical bonds naturallyor in vivo with residues outside of said ACE structure; and wherein saidnature covalent chemical bonds can be artificially, specifically andprecisely cleaved by designed chemical bond-specific hydrolysis insamples and sample preparations to create (the new terminals) and/orexpose said hidden ACE structure for antibody detection.

In another embodiment, the invention provides methods of using ACEantigen to make antibodies including, but not limited to, polyclonal,monoclonal, bi-specific, recombinant, humanized, antibody-likemolecules, and the like.

In a further embodiment, the invention provides methods of detecting ACEstructures in samples and sample preparations, wherein ACE in a sampleis poorly accessible or unrecognizable by antibodies, and thus must beartificially created (with new terminals) and/or exposed specificallyand precisely, rather than randomly or accidentally, by residue chemicalbond-specific hydrolytic enzymes or agents; wherein said hydrolyticenzymes and agents are specifically selected and should be mostly thesame, but may also be occasionally different, with the one(s) used forthe hydrolysis-guided ACE antigen design; wherein artificially,specifically and precisely creating (new terminals) and/or exposinghidden ACE antigens can be carried out in any samples or samplepreparations including, but not limited to, in vivo or in vitro, inwhole or part of biological bodies or organisms, in isolated organs ororganelles, in tissues or tissue sections (with or without fixation), inisolated or cultured cells, in tissue or cell lysates, in body fluids orcell culture medium, as well as in biochemical assay mixtures, onWestern blot or chromatographic membranes or any supporting matrices orsurfaces, in chromatographic and centrifuge fractions, in cellular orsubcellular fractions, and the likes. Examples are given below in FIGS.1-13.

In additional embodiment, the inventive ACE methods can be used toreduce non-specific bindings in all antibody-based applications. Thisutility is owing to the fact that the chemical bond-specific hydrolysisis able to specifically and precisely create and/or expose the ACEstructures, while breaking up other structures/epitopes that areotherwise able to bind to the ACE antibody non-specifically.

The inventive ACE methods, antibodies, reagents, immunoassays and kitscan be used in all antibody-based applications, including, but notlimited to, Western blot, immunocytochemistry, immunofluorescence,immunoelectron microscopy, immunoprecipitation, flow cytometry,Enzyme-Linked Immuno-Sorbent Assay (ELISA), peptide array, antibodyarray, or any other types of immunoassays, and the like.

The inventive ACE methods, antibodies, reagents, immunoassays and kitscan be employed in a number of areas, including, but not limited to: (a)determination of specific conjugation sites and their sequences, (b)monitoring changes in molecule-to-molecule conjugation sites in vivo andin vitro, (c) diagnosis of diseases, (d) determination of molecularconjugation enzyme and chemical agent activities, (e) determination ofmolecular conjugation enzyme modulator or cofactor activities, (f)development of therapeutic agents for treatment of diseases, and (g) thelike.

A few examples of diagnosing diseases with the ACE methods, antibodies,reagents, immunoassays, and kits may include, but are not limited to:(a) lysosomal storage diseases by assaying LC3II-related autophagyactivities; (b) different types of cancers by assaying thecancer-specific core-fucosylated proteins; (c) neurological,neurodegenerative, and conformational diseases, as well as heartdisease, muscle diseases (e.g., dermatomyositis), alcoholic liverdisease, stroke and diabetes by assaying cell-, tissue-, ororgan-specific or disease-specific macromolecule-to-macromoleculeconjugation sites including, but not limited to, ubiquitin andubiquitin-like conjugates, transglutaminase-mediated proteincross-links, telomeric aggregates, advanced glycation adducts, and thelike; (d) diseases-related biomarkers, such as aberrant p53ubiquitination-, sumoylation-and neddylation, and (e) the like.

The ACE methods, antibodies and reagents can be used in therapeuticapplications including, but not limited to, those related to abnormalmacromolecule-to-macromolecule conjugation.

The ACE methods, antibodies, reagents, immunoassays, and kits can beused in the area of biomaterials such as collagen-like biomaterials.

The ACE methods, antibodies, reagents, and immunoassays can be assembledto kits for research, diagnostic and therapeutic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

All hidden antigens designed below are derived from the commonstructures of either linear L1-L2 ------Ln or branchedLn---L2-L1(-S1-S2---Sm)-L1′-L2′---Lm′ ACE structures described above inthe section (SUMMARY OF THE INVENTION). In exemplary ACE structuresdescribed in this invention, the single capital letters representone-letter standard amino acid abbreviations. For instance, G=glycine,K=lysine, Q=glutamine, etc. The bold/italic (K) represents theconjugation site lysine. KLH all together is the abbreviation forKeyhole Limpet Hemocyanin, which is one of the most commonly usedimmunogenic carriers for antibody production. The dash “-” representscovalent chemical bond conjugation. The peptide sequence inside thebrackets “( )” often represents the branched monomers or oligomersincluding, but not limited to amino acid(s)/peptide(s), lipid(s),saccharide(s), nucleic acid(s), and the likes.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. A conjugation site-specific hidden antigen can be designed bythe ACE methods in different ways depending on which hydrolyticenzyme(s) or agent(s) will be used for detection. The following areexemplary designs of SUMO-to-protein conjugation site-specific hiddenACE antigens. Sequences in FIG. 1 are branched peptides, and no SEQ IDNOS are assigned to these branched sequences and other branchedsequences throughout this application.

FIG. 1A: A GluC-cleaved ACE antigen is designed asGVK(GGTQQQFVDITDC-KLH) TE, and can be used to make the SUMO-to-SUMO K11conjugation site-specific antibody. After treatment with GluC toartificially, specifically and precisely create and/or expose theGluC-cleaved ACE structure either in situ or ex situ in a sample, theSUMO-to-SUMO K11 conjugation sites can then be detected with the ACEantibody by all antibody-based methods.

FIG. 1B: A trypsin-cleaved ACE antigen is designed asEGVK(GGTQQQGGGC-KLH) TENN, and can be used to make the SUMO-to-SUMO K11conjugation site-specific antibody. After treatment with trypsin toartificially, specifically and precisely create (new terminals) and/orexpose the trypsin-cleaved ACE structure either in situ or ex situ in asample, the SUMO-to-SUMO K11 conjugation site can then be detected withthe ACE antibody by all antibody-based methods.

FIG. 1C: A GluC-cleaved ACE antigen is designed asKLH-CGGGALVAK(GGTQ)QE, and can be used to make the SUMO-to-NF-kappa-Bessential modifier (NEMO) K277 conjugation site-specific antibody. Aftertreatment with GluC to artificially, specifically and precisely create(new terminals) and/or expose the GluC-cleaved ACE structure either insitu or ex situ in a sample, the SUMO-to-NEMO conjugation site can thenbe detected with the ACE antibody by all antibody-based methods.

FIG. 1D: A GluC-cleaved ACE antigen is designed as RVK(GGTQQQGGGC-KLH)Eand can be used to make the Sumo2/3-to-Heat shock factor protein 1(HSF1) K298 conjugation site-specific antibody. After treatment withGluC to artificially, specifically and precisely create (new terminals)and/or expose the GluC-cleaved ACE structure either in situ or ex situin a sample, the SUMO2/3-to-HSF1 K298 conjugation site can then bedetected with the ACE antibody by all antibody-based methods.

FIG. 1E: A trypsin-cleaved ACE antigen is designed asLK(GGTQQQGGGC-KLH)K and can be used to make theSumo2/3-to-Hypoxia-inducible factor 1 alpha (HIF1alpha) K391 conjugationsite-specific antibody. After treatment with trypsin to artificially,specifically and precisely create (new terminals) and/or expose thetrypsin-cleaved ACE structure either in situ or ex situ in a sample, theSUMO2/3-to-HIF1alpha K391 conjugation site can then be detected with theACE antibody by all antibody-based methods.

FIG. 1F: A GluC-cleaved ACE antigen is designed as KLH-CGGGKLMFK(GGTQ)TEand can be used to make the Sumo1-to-p53 K386 conjugation site-specificantibody. After treatment with GluC to artificially, specifically andprecisely create (new terminals) and/or expose the GluC-cleaved ACEstructure in a sample, the SUMO1-to-p53 K386 conjugation site can thenbe detected with the ACE antibody by all immunological or antibody-basedmethods.

FIG. 2. Method 1a: A GluC-cleaved ACE antigen is designed asTFG-phosphotidylethanolamine (PE)-liposome and can be used to make theLC3II-specific antibody. The shaded area indicates the ACE structure.After treatment with GluC to artificially, specifically and preciselycreate and/or expose the GluC-cleaved ACE structure in a sample, theLC3II active form can then be specifically detected with the ACEantibody by all immunological methods. Method 1b illustrates how theTFG-PE ACE structure is artificially, specifically and precisely createdand/or exposed by GluC treatment in a lipid membrane sample preparation.

FIG. 2. Method 2a: A GluC-cleaved ACE antigen is designed as TFGGGGC-KLH(SEO ID NO: 14) and can be used to make the LC3II-specific antibody.After treatment with GluC to artificially, specifically and preciselycreate and/or expose the GluC-cleaved ACE structure in a sample, theLC3II active form can then be detected with the ACE antibody by allimmunological or antibody-based methods.

FIG. 2. Method 2b: A cyanogen bromide (CNBr)-cleaved ACE antigen isdesigned as VYASQETFGC-KLH (SEO ID NO: 15) and can be used to make theLC3II-specific antibody. After treatment with CNBr to artificially,specifically and precisely create and/or expose the CNBr-cleaved ACEstructure in a sample, the LC3II active form can then be specificallydetected with the ACE antibody by all antibody-based methods.

FIG. 2. Method 2c: A trypsin-cleaved ACE antigen is designed asDEDGFLYMVYASQETFGC-KLH (SEO ID NO: 16) and can be used to make theLC3II-specific antibody. After treatment with trypsin to artificially,specifically and precisely create and/or expose the trypsin-cleaved ACEstructure in a sample, the LC3II active form can then be detected withthe ACE antibody by all antibody-based methods.

FIG. 2. Method 3: A phospholipase D-cleaved ACE antigen is designed asKLH-CGGGSQETFG-ethanolamine (SEO ID NO: 17) and can be used to make theLC3II-specific antibody. After treatment with phospholipase D toartificially, specifically and precisely create and/or expose thephospholipase D-cleaved ACE structure in a sample, the LC3II can bedetected with the antibody by all immunological or antibody-basedmethods.

FIG. 2. Method 4: A phospholipase C-cleaved ACE antigen is designed asKLH-CGGGSQETFG-phosphoethanolamine (SEO ID NO: 17) and can be used tomake the LC3II-specific antibody. After treatment with phospholipase Cto artificially, specifically and precisely create and/or expose thephospholipase C-cleaved ACE structure in a sample, the LC3II can then bespecifically detected with the antibody by all immunological orantibody-based methods.

FIG. 3, Method 1: Two or more different glycoforms of the same proteinbackbone in a sample are digested withendo-beta-N-acetylglucosaminidase-D, F- and/or -H (Endo D/F/H) toartificially, specifically and precisely create and/or expose(monosaccharide) GlcNAc-and (disaccharide) Fuc-GlcNAc-glycoproteinhidden ACE structures. The hidden ACE structures can then be capturedfirst with a surface precoated with an antibody to a non-glycan portionof the protein (antibody 1). The fluorophor-1 -labeled GlcNAc-to-proteinconjugation site-specific antibody (antibody 2), and/or thefluorophor-2-labeld Fuc-GlcNAc-to-protein conjugation site-specificantibody (antibody-3) can bind to their corresponding ACE structurescaptured on the surface. After washing, the fluorophors can be detectedor imaged with a dual wavelength fluorometer.

FIG. 3, Method 2: Similarly, two or more different glycoproteins withthe same core-fucosylated N-glycan, e.g., glycoproteins-1 and -2 in asample, are digested with Endo D/F/H to artificially, specifically andprecisely expose the ACE hidden structures. The Fuc-GlcNAc-proteins-1and-2 in the samples are then captured with a surface precoated with theFuc-GlcNAc-protein glycoform-specific ACE antibody. After washing, afluorophor-1-labeled antibody against a non-glycan portion ofglycoprotein-1, and/or fluorophor-2-labeled antibody against anon-glycan portion of glycoprotein-2, are then added to label thecaptured glycoproteins. After washing, the fluorophors can then bedetected or imaged with a dual wavelength fluorometer.

FIG. 3, Method 3: N-glycan glycoproteins in a sample are digested withpeptide:N-glycosidae F (PNGase F) which cleaves the chemical bondbetween the innermost GlcNAc and asparagine residue of the proteins.This digestion removes all of the N-glycans from the protein. The PNGaseF-deglycosylated protein can still bind to the general antibody againstthe non-glycan portion of the protein, but cannot be recognized by the(monosaccharide) GlcNAc or (disaccharide) Fuc-GlcNAc glycoform-specificantibodies. This method can be used as a negative control of theglycoform-specific antibodies described above in Methods 1 and 2.

FIG. 3, Method 4: Western blot application of the glycoform-specificantibody. Samples from hepato-cellular carcinoma cell culture media weretreated with non-enzyme solution (Ctr), a mixture of endo-D/H, or withthe general PNGase-F (F), and then subjected to immunoblot analysis.Immunoblots were labeled with: (a) a general alpha-fetal protein (AFP)antibody, (b) the glycosylation site-specific antibody to disaccharideFuc-GlcNAc (core-fucosylation) AFP, and (c) the glycosylationsite-specific antibody to monosaccharide GlcNAc (no core-fucosylation)AFP. The general AFP antibody detects all glycosylated andde-glycosylated AFP bands (FIG. 3, Method 4a), the core-fucosylatedsite-specific AFP antibody labels only the core-fucosylated AFP (FIG. 3,Method 4b), and the monosaccharide GlcNAc site-specific AFP antibodylabels only the GlcNAc-AFP (FIG. 3, Method 4c).

FIG. 4. Exemplary rationales for the ACE hidden antigen design. Theubiquitin-to-ubiquitin K48 conjugation site peptide LIFAGK₄₈(GG)QLEDGRis concealed inside the ubiquitin-to-ubiquitin conjugate structure (FIG.4A). Furthermore, the conjugation site-specific ACE peptideLIFAGK₄₈(GG)QLEDGR within the intact K48 conjugate has no charged freeterminals and thus is less antigenic (FIG. 4B). The trypsin treatmentartificially, specifically and precisely: (i) creates three newantigenic free terminals, and (ii) opens up to expose the conjugationsite-specific ACE structure LIFAGK₄₈(GG)QLEDGR, and thus makes thishidden epitope more antigenic and accessible to antibody (FIG. 4C).

FIG. 5: Trypsin-cleaved ACE hidden antigens: (A) ubiquitin-to-HIF1alpha;and (B) ubiquitin-to-histone 2A, are designed as KLH-CGGGMVNEFK(GG)LELand LLPKK(GG)TESHHGGGC-KLH, respectively. These two antigens can be usedto make the ubiquitin-to-HIFlalpha and ubiquitin-to-histone 2Aconjugation site-specific antibodies, respectively. After treatment withtrypsin to artificially, specifically and precisely create and/or exposethe corresponding trypsin-cleaved hidden ACE structures in samplepreparations, the ubiquitin-to-HIFlalpha and ubiquitin-to-histone 2Aconjugation sites can then be detected with their corresponding ACEantibodies by all antibody-based methods.

FIG. 6. Trypsin-cleaved ACE hidden antigens are designed as: (1)KLH-CGGGAVVPC₁₃₃(palmitate)IK (SEO ID NO. 3) for making the C133palmitoylated caveolin-1 lipidation site-specific ACE antibodies; (2)liposome-Myristate-GSNK (SEO ID

NO. 4) for making p60c-src myristoylation site-specific ACE antibodies;(3) KLH-CGGGSGG₁₉₇(cholesterol)CF (SEO ID NO. 5) for making the Hedgehogcholesterol-conjugation site-specific ACE antibodies; and (4)KLH-CGGGKC₁₈₆(farnesol)VLS (SEO ID NO. 6) for making p21-Rasfarnesylation conjugation site-specific ACE antibodies. After treatmentwith trypsin to artificially, specifically and precisely create and/orexpose the corresponding trypsin-cleaved hidden ACE structures in samplepreparations, the lipidation-specific ACE structures can then bedetected with their corresponding ACE antibodies, respectively, by allantibody-based methods. Similarly, (also in FIG. 6) a LysN-cleaved orpeptidyl-Lys metalloendopeptidase-cleaved hidden ACE antigen is designedas KLH-CGGGKC₁₈₉(geranylgeranyl isoprene)LLL (SEO ID NO. 7) for makingp21-Rac1 (ras-related C3 botulinum toxin substrate 1)geranylgeranylation conjugation site-specific antibodies. Aftertreatment with LysN or peptidyl-Lys metalloendopeptidase toartificially, specifically and precisely create and/or expose the ACEstructure in sample preparations, the geranylgeranylated p21-Rac1conjugation site can then be specifically detected with the antibodiesby all antibody-based methods.

FIG. 7. Two trypsin-cleaved ACE hidden antigens are designed as: (1)GC-phosphoethanolamine-SGGGC-KLH (SEO ID NO: 8); and (2)TS-phosphoethanolamine-SGGGC-KLH (SEO ID NO: 9) for making theglycosylphosphatidylinositol (GPI)-to-prion protein (PrP), andGPI-to-glypican, conjugation site-specific antibodies, respectively.After treatment with trypsin to artificially, specifically and preciselycreate and/or expose the trypsin-cleaved ACE structures in samples orsample preparations, the PrP-to-GPI or glypican-to-GPI conjugation sitescan then be detected, respectively, with their corresponding ACEantibodies by all antibody-based methods.

FIG. 8. A trypsin-cleaved ACE antigen is designed as KLH-CGGGQK(DQLGK)TV(SEO ID NOS: 10-11) for making the transglutaminase-mediatedsynuclein-to-synuclein conjugation site-specific antibodies. Aftertreatment with trypsin to artificially, specifically and preciselycreate and/or expose the trypsin-cleaved hidden ACE structure in samplesor sample preparations, the synuclein-to-synuclein conjugation site canthen be detected with this ACE antibody by all antibody-based methods.

FIG. 9. Immunohistochemistry labeling of the ubiquitin-to-ubiquitin K48conjugation site-specific ACE structure in tissue sections. Tissuesections were treated without (FIG. 9A) or with (FIG. 9B) trypsin, andthen double-labeled with the ubiquitin-to-ubiquitin K48 ACE antibody andpropidium iodide (PI). PI stains nuclei in a red color, while theubiquitin-to-ubiquitin K48 conjugation site-specific antibody-labeledconjugation site is shown in a green color. In FIG. 9A, without trypsintreatment, the hidden ubiquitin K48 conjugation site ACE structure wasnot artificially, specifically and precisely created and/or exposed, andthus could not be labeled with the K48 ACE antibody in the green color.In comparison, in FIG. 9B, because the hidden ubiquitin K48 conjugationsite ACE structure were exposed artificially and specifically by trypsintreatment, the ubiquitin K48 conjugation site-specific antibody labeledthe hidden ACE structure in the green color. PI-stained nucleic acids inthe red color were not affected by trypsin treatment, and thus are seenin both trypsin-treated and-untreated tissue sections.

FIG. 10. Immunoblots of the ubiquitin-to-ubiquitin K48 ACE structure onWestern blot membranes. Equal volumes of tissue lysates with equalizedprotein concentration were subjected to Western blot analysis. TheWestern blot membranes were treated either with (FIG. 10A) or without(FIG. 10B) trypsin. A: The ubiquitin-to-ubiquitin K48 conjugation siteACE structure was labeled with the conjugation site-specific antibodybecause the hidden ACE structure was artificially, specifically andprecisely created and/or exposed with trypsin treatment. B: The hiddenubiquitin-to-ubiquitin K48 conjugation site ACE structure was notlabeled with the ACE antibody because, without trypsin treatment, thehidden conjugation site ACE structure remained hidden, and thus was notartificially, specifically and precisely created and/or exposed onWestern blot membrane.

FIG. 11. Immunoblots of hidden ACE antigen designs and detections. Equalprotein concentrations and volumes of tissue lysates were subjected toWestern blot analysis. The Western blot membranes were treated eitherwithout (−) or with (+) trypsin (FIG. 1A-D), or GluC (FIG. 1E).According to the ACE methods, the selection of either trypsin or GluC inthis situation must be based on the protease information used for theACE antigen design. The ubiquitin-to-protein conjugation site ACEstructures were designed as CIMESFK(GG)NMVPQQALVIR for Na⁺/K⁺ ATPase orATP1A1 (FIG. 1A); CHLNK(GG)MQNHGYENPTYK for Amyloid beta precursor-likeprotein 2 or Ap1p2 (FIG. 1B); CLHEDPPQK(GG)PPR for amyloid beta proteinprecursor intracellular domain associated protein-1b or AIDA-1b (FIG.1C); and CYQLFEELGK(GG)GAFSVVR (K21) for calcium/calmodulin dependentkinase II or CaMKII (FIG. 1D). These are trypsin-digested segments ortheir truncated forms. The SUMO2/3-to-protein conjugation ACE structurewas designed as CGGKPKEGVK(GGTQQQ)TE (FIG. 1E), which is a GluC-digestedsegment. Without trypsin or GluC treatment (−), the specific proteinbands were hidden and thus could not be or poorly detected with theircorresponding antibodies. In contrast, after trypsin or GluC treatment(+) to artificially, specifically and precisely create and/or expose theACE structures, both the conjugated (arrowheads), and non-conjugatedprotein hidden bands (arrows) can be detected with their correspondingantibodies. * indicates the nonspecific bands because their molecularsizes do not match with the proteins of interests.

FIG. 12. Immunohistochemistry of tissue sections double-stained with theubiquitin-to-histone K120 conjugation site-specific ACE antibody (green)and PI (red). Tissue sections were treated without (A) or with (B)trypsin. A: Without trypsin treatment, the hidden ubiquitin-to-histoneK120 ACE structure was not artificially, specifically and preciselycreated and/or exposed. Therefore, only PI-stained nuclei in the redcolor are seen. B: Both PI-stained red nuclei, and theubiquitin-to-histone K120 conjugation site-specific antibody-labeledgreen ACE structure are seen, because the hidden K120 ACE structure wasartificially, specifically and precisely created and/or exposed bytrypsin treatment.

FIG. 13. Immunoblots of the ubiquitin-to-histone K120 conjugation site.Western blot membranes were treated either with trypsin (A) or with thebuffer only (B, no trypsin). After washing, membranes were labeled withthe ubiquitin-to-histone K120 conjugation site-specific rabbit crudeantiserum. After washing, membranes were labeled with aperoxidase-labeled secondary antibody and developed with an enhancedchemiluminescence (ECL) Western blot system. A: The hiddenubiquitin-to-histone conjugation site ACE structure was specificallylabeled with the conjugation site-specific antiserum because the hiddenACE structure was artificially, specifically and precisely createdand/or exposed with trypsin treatment. Arrow indicates themonoubiquitin-to-histone band. B: The hidden ubiquitin-to-histoneconjugation site ACE structure was not labeled with the antiserumbecause, without trypsin treatment, the hidden ACE structure was notartificially, specifically and precisely created and/or exposed.Therefore, the antiserum-labeled bands on Western blot membrane arelikely non-specific bindings.

FIG. 14. An antibody-based time-resolved fluorescence resonance energytransfer (TR-FRET) assay design. The human double minute (Hdm2) oncogeneE3 ubiquitin ligase catalyzes ubiquitination of p53 at a lysine residueK370. The conjugation site-specific antibody to the ubiquitin-to-p53K370 site can be made with KLH-CGGGSSHLK₃₇₀(GG)SK) (no SEQ ID NO isassigned to this branched sequence). The assay mixture consists of abiotinylated p53 peptide substrate (biotin-CGGGSSHLKSK), Hdm2, ATP, K48Rubiquitin, E1, and E2 (UbcH5c). After incubation, a stop buffer (e.g.,40 mM EDTA) containing detection mix (ULight-streptavidin and theEuropium Cryptate-labeled ubiquitin-to-p53 conjugation site-specificantibody) is then added. The p53 E3 ligase activity can then be measuredwith a time-resolved fluorometer at excitation of 320 nm and emission665 nm.

FIG. 15. A-B: Examples of Protein Glycosylation ConjugationSite-Specific ACE Structures. In the following sequences, the ACEstructure with branch (GlcNAc or Fuc-alpha1,6-GlcNAc) conjugation iscreated in a sample preparation by endoglycosidases treatment. In thiscase, only the polysaccharide chain is cleaved, while the proteinpolypeptide chain is not cleaved. In the hidden antigen case, however,the sample preparation also needs to be treated with the designatedhydrolytic enzyme or hydrolytic agent in order to create and/or exposethe hidden antigen for antibody detection.

FIG. 15A. Protein Glycosylation Sequence ID NOS: 1 and 18-28.

FIG. 15B. Protein Glycosylation Sequence ID NOS: 29-39.

FIG. 16. A-E: Examples of Protein Sumoylation Conjugation Site-SpecificACE Structures. In the following sequences, the branch structure (GGTQ)is derived from any isoform of SUMO proteins including, but not limitedto, SUMO1 and SUMO2/3. The ACE structure with an E (=glutamate) residueat the C-terminal is created in a sample preparation by GluC treatment,whereas the ACE structure with the C-terminal K (=lysine) or R(=arginine) residue is created by trypsin treatment. There is anexception in the situations that there is no E, K or R residue betweenthe conjugation site and the protein C-terminal.

FIG. 16A. Part A of Sumoylation Conjugation Site-Specific ACEStructures.

FIG. 16B. Part B of Sumoylation Conjugation Site-Specific ACEStructures.

FIG. 16C. Part C of Sumoylation Conjugation Site-Specific ACEStructures.

FIG. 16D. Part D of Sumoylation Conjugation Site-Specific ACEStructures.

FIG. 16E. Part E of Sumoylation Conjugation Site-Specific ACEStructures.

FIG. 17. A-D: Examples of Protein Ubiquitination ConjugationSite-Specific ACE structures. In the following sequences, the ACEstructure with branch (GG) conjugation is created in a samplepreparation by trypsin treatment, so that the C-terminal is either a K(=lysine) or R (=arginine) residue. The ACE structure with branch(GGRLRLVLHLTS) conjugation is created by GluC treatment, and, as aresult, the C-terminal is E (=glutamate). There is an exception in thesituations that there is no E, K or R residue between the conjugationsite and the protein C-terminal, or that the sequence between theconjugation site and the next E, K or R residue is too long to exposethe hidden antigen.

FIG. 17A. Part A of Ubiquitination Conjugation Site-Specific ACEstructures.

FIG. 17B. Part B of Ubiquitination Conjugation Site-Specific ACEstructures.

FIG. 17C. Part C of Ubiquitination Conjugation Site-Specific ACEstructures.

FIG. 17D. Part D of Ubiquitination Conjugation Site-Specific ACEstructures.

FIG. 18. Examples of Protein Neddylation Conjugation Site-Specific ACEStructures. In the following sequences, the ACE structure with branch(GG) conjugation is created in a sample preparation by trypsintreatment, so that the C-terminal is either a K (=lysine) or R(=arginine) residue. The ACE structure with branch (GGRLRLVLHLTS)conjugation is created by GluC treatment, and, as a result, theC-terminal is E (=glutamate). There is an exception in the situationsthat there is no E, K or R residue between the conjugation site and theprotein C-terminal.

FIG. 19. SEQ ID NOS 40-52 are examples of Linear Hidden ACE Structures.In the following sequences, the linear hidden ACE structures areprotein-to-protein non-covalent binding sites with the commonhydrophobic motif (as indicated with bold/italic letters). They areeither trypsin (with the terminal K or R), or GluC (with the terminal E)cleaved protein segments.

DETAILED DESCRIPTION

The invention summarized above may be better understood by referring tothe following description, which should be read in conjunction with theaccompanying claims and drawings in which like reference numbers areused for like parts. This description in which some examples of theembodiments of the inventions are shown, is to enable one to build anduse an implementation of the invention, and is not intended to limit theinvention, but to serve as a particular example thereof. Those skilledin the art should appreciate that they may readily use the conceptionand specific embodiments disclosed as a basis for modifying or designingother methods and systems for carrying out the same purposes of thepresent invention. Those skilled in the art should also realize thatsuch equivalent methods, designs, constructs, antibodies, kits, samples,and cell lines do not depart from the spirit and scope of the inventionin its broadest form.

Definitions

As used herein, the term “detection” or “detecting” is interchangeablewith discovering, uncovering, finding, recognizing, revealing,determining, examining, measuring, and the like.

As used herein, “in situ” refers to a phenomenon or event occurred inexactly the original location, both in vivo and in vitro, including butnot limited to, in whole or part of biological bodies or organisms, inisolated organs, cells, and organelles, in tissues or tissue sections(with or without fixation), in isolated or cultured cells, in bodyfluids or cell culture media, as well as on Western blot membranes, andany supporting matrices or surfaces, in chromatographic and centrifugefractions, in reaction mixtures, and the like.

As used herein, “ex situ” is the opposite of “in situ”, and refers to aphenomenon or event that does not occur in the original place both invivo and in vitro.

As used herein, the term “hidden antigen” is often interchangeable with“hidden hapten or segment” or “ACE structure”, and refers to an antigenepitope/segment/structure that, in its intact or natural form, is lessantigenic and/or poorly accessible to large molecules including, but notlimited to, antibodies. For example, an ACE structure may be amacromolecule-to-macromolecule conjugation site, or a segment normallylocated inside its parent macromolecule, or may be covered by othersurrounding molecules/structure(s)/cell membranes either in situ or exsitu, and thus is poorly or not accessible to antibodies.

As used herein, the term “macromolecule” refers to a polymeric moleculewith more than 2 same or different units, either in a linear or branchedsequence, including, but not limited to, polypeptides, polysaccharides,lipids or phospholipids, and nucleic acids, poly(ADP-ribose), or anycombinations of the above.

As used herein, the term “macromolecule-to-macromolecule conjugation”refers to that process in which one macromolecule conjugates to anothersame or different macromolecule via a covalent linkage.

As used herein, the term “carbohydrate” is interchangeable with the term“saccharide”, typically referring to either polymeric or monomeric sugarmolecules.

As used herein, the term “conjugation site” refers to the site where acovalent linkage is formed between two macromolecules, mostlyterminal-to-sidechain branched conjugations, and occasionally molecularhead-to-tail linear conjugations.

As used herein, the term “conjugation site-specific hapten” refers to asegment that contains a macromolecular conjugation site, which may needto be linked to an immunogenic carrier in order to become a completeantigen.

As used herein, the term “conjugation site-specific antibody” refers toantibody that can specifically recognize amacromolecular-to-macromolecular conjugation site derived from bothmacromolecular moieties.

As used herein, the term “sidechain” refers to a chemical group that isattached to or branches from a core part of the molecule called the“mainchain” or backbone. In polymers, side chains extend from a backbonestructure.

As used herein, the term “hydrolytic enzyme” refers to proteases,glycosidases, lipases or phospholipases, nucleases, and the like, whichare currently known or will be identified in the future and are capableof cleaving particular chemical bonds in macromolecules in asite-specific manner.

As used herein, the term “agent”, may be interchangeable with“hydrolytic agent” or “chemical agent”, and refers to chemicals or anyother non-biological materials that are currently known or will beidentified in the future, and are capable of cleaving particularchemical bonds in macromolecular backbones in a site-specific manner.Hydrolytic agents may include, but are not limited to,2-nitro-5-thiocyanobenzoic acid (NTCB) +Ni that cleaves the peptide bondat cysteine loci (Degani and Patchornik, 1974); cyanogen bromide (CNBr)that cleaves at methionine loci; BNPS-skatole[2-(2-nitrophenylsulfenyl)-3-methylindole] that cleaves at tryptophanloci; and formic acid that cleaves at aspartate loci in proteinbackbones.

Hydrolytic proteases and agents include, but are not limited to, Arg-Cproteinase, Asp-N endopeptidase, Asp-N endopeptidase+N-terminal Glu,BNPS-Skatole, caspasel, caspase2, caspase3, caspase4, caspase5,caspase6, caspase7, caspase8, caspase9, caspase10, chymotrypsin,clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa,formic acid, glutamyl endopeptidase, granzymeB, hydroxylamine,iodosobenzoic acid, LysC, LysN, NTCB (2-nitro-5-thiocyanobenzoic acid),pepsin, proline-endopeptidase, proteinase K, staphylococcal peptidase I,tobacco etch virus protease, thermolysin, thrombin, trypsin, and thelike.

The chemical bond-cleaving site specificities of hydrolytic enzymes oragents can be found in publicly accessible databases including, but notlimited, to Swiss-Prot ExPASy and the National Center for BiotechnologyInformation.

Glycosidases include, but are not limited to, exoglycosylase,endoglycosylase, any combination of exoglycosylase and endoglycosylase,and/or sialidase, fucosidase, mannosidase, galactosidase, xylosidase,and the like.

Lipases include, but are not limited to, triglyceride lipase, pancreaticlipase, lysosomal lipase, hepatic lipase, hormone-sensitive lipase,endothelial lipase, lingual lipase, and the like.

Phospholipases include, but are not limited to, phospholipase A1,phospholipase A2, phospholipase B, phospholipase C, phospholipase D,GPI-phospholipase C, GPI-phospholipase D, and the like.

Macromolecular conjugation enzymes include but are not limited to E3ubiquitin-protein ligase; UBL ligase, carbohydrate transferase, poly(ADP-ribose) polymerase, fatty acyl transferase; autophagy-relatedprotein ligase [e.g., autophagy-related gene (ATG) 3 and ATG10],sumoylation ligase, neddylation ligase, transglutaminase, and the like.

Macromolecular de-conjugation enzymes may include, but are not limitedto, deubiquitination enzymes (DUBs) and UBL proteases; desumoylationenzymes or SUMO proteases; deglycosylation enzymes, lipases,phospholipases, poly (ADP-ribose) glycohydrolases (PARG), and the like.

Enzymes used in the invention may be natural, recombinant or chemicallysynthesized. They may be substantially pure, partially purified, orpresent in a crude biological sample.

As used herein, the term “organism” refers to all cellular life-formsincluding, but not limited to, prokaryotes and eukaryotes, non-cellularlife-forms, and nucleic acid-containing entities including, but notlimited to, bacteriophages and viruses.

As used herein, the term “sample or sample preparation” refers to acollection of inorganic, organic or biochemical molecules either in apure or mixture form, either in nature (e.g., in a biological-or otherspecimen) or artificial type, either in heterogeneous or homogeneousform, either in isolated, partially isolated or non-isolated form, oreither in solution or in a form immobilized or semi-immobilized on anysupporting materials including but not limited to electrophoresis matrix(e.g., gel or capillary), Western blot membrane (e.g., nitrocellulosemembranes), agarose support (e.g., gel or bead), nano particles, anysupporting surface, cell culture plates, multiplex beads, orchromatographic supporting matrix, sucrose gradient medium. “Sample”further refers to a biological sample.

As used herein, the term “organism” refers to all cellular life-forms,including but not limited to prokaryotes and eukaryotes, as well asnon-cellular life-forms, nucleic acid-containing entities, including butnot limited to bacteriophage and viruses.

As used herein, the terms “biological sample” refer to a collection of awhole organism or a subset of its tissues, cells or component parts(e.g. body fluids, including but not limited to blood, mucus, lymphaticfluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid,amniotic cord blood, urine, vaginal fluid and semen). “Biologicalsample” further refers to a homogenate, lysate, subcellular fraction orextract prepared from a whole organism or a subset of its tissues, cellsor component parts, or a portion thereof “Biological sample” also refersto sample preparations including but not limited to on electrophoreticand chromatographic gels, on Western, Southern, and Northern blotmembranes, in isolated organelles, and in separated fractions.

As used herein, the term “tissue section” refers to a thin sliceprepared from a whole organism or a subset of its tissues, cells orcomponent parts (e.g. body fluids, including but not limited to blood,mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva,amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).

As used herein, the term “conjugation site-specific antibody” furtherrefers to one or more antibodies, and also referred to asimmunoglobulins. Conjugation site-specific antibody may be natural orpartially or wholly produced artificially, e.g. recombinant, or antibodyfragments. A conjugation site-specific antibody may be monoclonal orpolyclonal, humanized, bispecific, and heteroconjugate antibodies, aswell as antibody-like binding partners (e.g., phage displayantibody-like fusion protein). Conjugation site-specific antibody may bemade in all immunoreactive animals or organisms including but notlimited to rabbit, rat, mouse, sheep, horse and donkey. The antibodymay, in some cases, be a member of one, or a combination immunoglobulinclasses, including: IgG, IgM, IgA, IgD, and IgE, as well asantibody-like molecules.

As used herein, the term “de-conjugation site-specific antibody” refersto one or more antibodies that recognize the conjugation site in thenon-conjugated form.

As used herein, the term “pan antibody” refers to one or more antibodiesthat recognize epitopes that are not located on the conjugation site.

The conjugation site-specific haptens or antigens may be used to selectits binding partners by, for example, phage display or yeast display.The haptens or antigens include, but are not limited to, any chemicalmonomers or polymers, amino acids or peptides, carbohydrates, lipids orphospholipids, nucleotides, poly (ADP-riboses), and the like.

As used herein, the term “antigenicity” refers to the antigen capacityto stimulate the production of antibodies and the capacity to react withantibodies.

As used herein, the term “primary antibody” refers to antibody raisedagainst an epitope of interest. The epitope can be a protein, peptide,carbohydrate, lipid, phospholipid, nucleic acid, any combination of theabove, or any other macromolecules.

As used herein, the term “secondary antibody” refers to an antibody thatbinds to primary antibodies or antibody fragments. They are typicallylabeled with measurable probes for detection, purification, or cellsorting applications.

As used herein, the term “immunoassay” refers to any antibody-basedmeasurement of the content of any substance in a sample. The presence ofantigen and/or antibody can be assayed. The most common method is tolabel either antigen or antibody with any suitably detectable materialsincluding, but not limited to, enzymes, radioisotopes, magnetic orfluorescent labels, or nanoparticles.

As used herein, the term “Western blot” or its interchangeable term“immunoblot” refers to an analytical method for detection of proteins ormodified proteins in a sample. It uses gel electrophoresis to separatemolecules in a sample. The separated molecules are then transferred to amembrane (typically nitrocellulose) that can hold the macromolecules,where such macromolecules of interest can be detected specifically withantibodies.

As used herein, the term “Enzyme-Linked ImmunoSorbent Assay” or “ELISA”refers to any method of detecting the presence and level of an antibodyor an antigen in a sample. There are several variants of ELISA,including, but not limited to, sandwich ELISA, competitive ELISA,indirect ELISA, ELISA Reverse and the like. The most common procedure isto coat an antibody or antigen onto a surface, and then to add moleculesof interest (antigen or antibody) to the precoated surface so that anantibody to antigen complex can form. The tagged antibodies or antigens,or the added secondary antibody with a detectable tag, can then bedetected with a readout system.

As used herein, the term “immunohistochemistry” commonly refers to amethod of antibody-based localization of antigens in a sample, commonlyin a tissue section. An antibody to antigen interaction can bevisualized by microscopy at the cellular level via any detectable meansincluding, but not limited to, antibodies tagged by fluorophors,chromospheres or luminescence, or any detectable tags with anycombinations of the above, including, but not limited to, peroxidase andits variants, chemiluminescence and its variants, and fluorescentmolecules such as fluorescein isothiocyanate (FITC), Texas Red,rhodamine (TRITC), coumarin, cyanine, Alexa Fluors and the DyLightFluors, and their derivatives.

As used herein, the term “immunocytochemistry” is often interchangeablewith immunohistochemistry. Immunocytochemistry emphasizes a method ofusing antibodies to detect specific antigens at the cellular level.Immunocytochemistry may differ somewhat from immunohistochemistry inthat it is often performed on samples of intact cells, whereasimmunohistochemical samples are usually on tissue sections.

As used herein, the term “immunoprecipitation” refers to a technique ofantibody precipitating its antigen molecule out of mixture samples. Thisprocess is often used to isolate and concentrate a particular antigen orantigen complex from other molecules in a sample. Immunoprecipitationoften requires coupling antibody-antigen complexes to a solid supportsubstance in the procedure for separation of antibody-antigen complexesfrom other molecules in a sample.

As used herein, the term “co-immunoprecipitation” refers toimmunoprecipitation of intact antigen complexes.

As used herein, the term “flow cytometry” refers to a method ofcounting, examining, and sorting particles suspended in a stream offluid. It allows simultaneous multi-parametric analysis of the physicaland/or chemical characteristics of single cellular particles flowingthrough an optical and/or electronic detection apparatus.

“Post-translational modification” refers to any chemical modification ofa polypeptide chain during and after it is synthesized, including butnot limited to phosphorylation, acylation, acetylation, methylation,sulfonation, glycosylation, lipidation, prenylation, isoprenylation,ubiquitination, biotinylation, formylation, citrullination,myristoylation, sumoylation, gamma carboxylation, ADP-ribosylation,amidation, covalent attachment of a moiety including but not limited toflavin, heme, nucleotide or nucleotide derivative, phosphotidylinositol,cyclization, demethylation, formation of covalent cross-links includingbut not limited to transglutaminase-mediated protein cross-linking,formation of pyroglutamate, GPI anchor formation, hydroxylation,iodination, oxidation, proteolysis processing, racemization,selenoylation, and the like. A post-translational modification may alsoinvolve cleavage of the polypeptide chain, proteolytic processing, theformation of disulfide bonds, and the like.

As used herein, the term “glycosylation” refers to the enzymatic ornon-enzymatic process that links at least one saccharide moiety toproteins, lipids or other macromolecules. The majority of proteinssynthesized in the rough endoplasmic reticulum (ER) undergoglycosylation. Glycosylation also occurs in the cytoplasm and nucleusincluding, but not limited to, the O-GlcNAc (N-acetylglucosamine)modification. Cells can produce several classes of glycans including,but not limited to: N-linked glycan attached to the amide nitrogen ofasparagine sidechain of a polypeptide; O-linked glycan attached to thehydroxyl oxygen of serine and threonine sidechain of a polypeptide;glycosaminoglycans attached to the hydroxyl oxygen of serine in apolypeptide; glycolipids in which glycans are attached to ceramide andhyaluronan, and GPI anchors which link proteins to lipids via glycanlinkages.

“Core fucosylation” refers to the linkage of a fucose residue to thecore N-acetylglucosamine via alpha1-6 linkage. All N-linked glycanstructures have a common structure, referred to as the core, containingthree mannose and two N-acetylglucosamine residues.

As used herein, the term “glycoform” refers to a group of proteinshaving an identical backbone amino acid sequence but differentcarbohydrate moieties.

As used herein, “artificially cleaved epitope or ACE” refers to anepitope that is artificially cleaved for creating more antigenic andaccessible epitope to antibody for detection or forming antibody-to-ACEcomplex purpose.

The “Artificially Cleaved Epitope” or “ACE” does not include naturallycleaved antigens in vivo.

As used herein, “hydrolysis-guided ACE design or ACE design” refers toantigen design methods that use an artificially cleaved epitope (ACE) asa hapten or antigen. The ACE is not naturally accessible to antibody orantibody-like molecules but can be artificially, specifically andprecisely created and/or exposed in a sample by the chemicalbond-specific hydrolysis of macromolecules.

As used herein, “hydrolysis-guided ACE antibody production” isinterchangeable with “ACE antibody production” refers to making antibodywith an ACE hapten or antigen designed by the ACE design methods.

As used herein, “residue” and “monomer” (of macromolecules) is ofteninterchangeable, refers to a specific unit within polymeric chains ofpeptides, polysaccharides, lipids, nucleic acids, poly(ADP-ribose), andthe like.

As used herein, “create or expose” (of ACE antigen) is ofteninterchangeable.

As used herein, the term “create” refers to artificially creating ACEstructures with free terminals in samples or sample preparations forantibody detection.

As used herein, “ACE methods” refers to a group of collective techniquesincluding, but not limited to: (i) ACE design, (ii) making ACE antibody,(iii) ACE exposure and detection either in situ or ex situ in a sample,and (iv) ACE method applications. The ACE methods require to use: (a)structural and sequence information of molecular conjugates or linearhidden antigen, (b) chemical bond-cleaving specificities and propertiesof hydrolytic enzymes and agents, and (c) ACE exposure and detectionmethods.

1. Methods of Design Ace Antigens

Extensive efforts on generation of macromolecule-to-macromoleculeconjugation site-specific antibodies by conventional antibody methodshave been proven futile (Matsumoto et al., 2008). Our recent studysuggests that these unsuccessful attempts are mainly because mostmacromolecule-to-macromolecule conjugation sites are either poor antigenor hidden on the inside of the conjugated macromolecules, and thuspoorly or not accessible to antibody binding (see FIG. 4 as a example).This invention provides novel methods to solve these problems. Thesemethods are generally and collectively referred to as the“hydrolysis-guided ACE methods” or simply “ACE methods”, including, butnot limited to, all or part of these steps: (i) selecting a specifichydrolytic enzyme or chemical agent-cleaved conjugation site-specific ora hidden ACE sequence or its truncated form; (ii) the ACE structure is anon-cleaved segment of an intact macromolecule in vivo, and is eitherpoorly antigenic, or inaccessible by antibody, but can be artificially,specifically and precisely created and/or exposed by the residuechemical bond-specific hydrolytic enzyme, agent or their combinations,(iii) conjugate the ACE hapten to an immunogenic carrier for making it acomplete antigen, (iv) a spacer including but not limited to GGG(glycine-glycine-glycine) may sometimes be added to increase theflexibility of the ACE, (v) use the complete antigen to make theantibodies or antibody-like molecules or binding partners, (vi) removeantibodies to non-conjugation site by negative absorption with thenon-conjugated linear peptide(s), (vii) create and/or expose the ACEartificially, specifically and precisely, rather than randomly oraccidentally, either in situ or ex situ in any sample preparations bythe specifically designated chemical bond-specific hydrolytic enzyme ormeans, and (viii) detect or image the ACE structure in samplepreparations by antibody-based methods.

Comprehensive information about macromolecule-to-macromoleculeconjugation sequence structures, and residue chemical bond-specifichydrolytic enzymes and chemical agents, can be found in the literatureand publically accessible databases including but not limited toNational Center for Biotechnology Information at www.ncbi.nlm.nih.gov/and ExPASy at www.expasy.ch/.

1.1. Macromolecular conjugation site-specific ACE structure design. TheACE methods for designing an ACE structure are based on information of:(a) the molecular structure and ACE sequence, (b) availability andproperty of chemical bond-specific hydrolytic enzymes or agents, and (c)methods of sample analyses (see FIGS. 1-14). Therefore, the ACE methodsare highly predictable, reliable and sensitive, as demonstrated in FIGS.1-14. Generally, the ACE structure at a hiddenmacromolecule-to-macromolecule conjugation site has all or part of thesecharacters: (i) comprises of a shorter piece (usually 1-6 monomers)derived from one macromolecule and a longer piece (usually 3-100monomers) derived from another macromolecule; (ii) must be antigenic;and (iii) must be poorly exposed and/or poorly antigenic naturally or invivo, and thus must be artificially, specifically and precisely created(free terminals) and/or exposed in samples and sample preparations bychemically engineered means, including, but not limited to, by usingchemical bond-specific hydrolytic enzymes, agents, or theircombinations.

A macromolecule-to-macromolecule conjugation site-specific ACE structurecan be described as: Ln------L2 -L1 (-S1 -S2 ------Sm)-L1′-L2′------Lm′,wherein L(# or #′)s stand for residues and their numbers of one (e.g.,long) macromolecular segment which has a branched covalent conjugationwith a second macromolecule at L1; wherein S(#)s stand for residues andtheir numbers of the second (e.g., short) macromolecular segment inwhich S1 is covalently conjugated to L1; wherein L1, L2 etc. are countedtoward one direction from the conjugation site (e.g., to the N-terminaldirection of a polypeptide); wherein L1′, L2′ etc. are counted towardthe opposite direction from the conjugation site; wherein Sl, S2 etc.are counted toward the third or branched direction from the conjugationsite; wherein the conjugation site is located between L1 and S1; whereinn, m and m′ are residue numbers continuously counted from the L1, L1′and S1, respectively; wherein Ln, Lm′, or Sm are not free ends naturallyand have natural chemical bonds with residues outside of the ACEstructure; and wherein the nature chemical bonds between Ln, Lm′ or Smand their ACE outside residues can be artificially, specifically andprecisely cleaved by the chemical bond-specific hydrolysis in any samplepreparations to expose the ACE structure(s) for detection.

Examples of the specific ACE structures are listed in various sectionsof this application, as well as in FIGS. 1-8.

By using the inventive ACE methods, we have successfully made severalconjugation site-specific ACE antibodies that are highly specific tomacromolecular conjugation sites in tissue sections or Western blotmembranes (see FIGS. 3 and 9-13).

1.2. The ACE antigen design for detecting linear hidden antigen. Inaddition to macromolecular conjugation site, the inventive ACE methodscan also be used to detect any types of hidden antigens in samples orsample preparations, and have an advantage over the conventional antigendesign and detection methods. This is because the chemical bond-specifichydrolysis strategy can artificially, specifically and precisely createand/or expose simpler and/or more charged terminal antigenic epitope(s).

The non-branched linear hidden ACE also has a general structure: L1 -L2------Ln, wherein L1-L2 ------Ln is a single polymeric chain (e.g.,polypeptide, polysaccharide, lipid, polynucleic acids, or theircombinations), and either poorly antigenic or cannot be accessible inits original nature form by antibody, and thus must be artificially,specifically and precisely created and/or exposed by residue chemicalbond-specific hydrolysis in situ or ex situ in sample preparations;wherein L(#)s stand for residues of the hidden antigen; wherein L1and/or Ln are not free ends naturally or in vivo and have chemical bondsnaturally with residues outside of the ACE structure; wherein the naturechemical bonds can be artificially, specifically, and precisely cleavedby residue chemical bond-specific hydrolysis in any types of samplepreparations to create and/or expose the ACE structures for detection.An example is given in FIG. 2.

1.3. The ACE antigen design for reducing non-specific bindings. The ACEmethods can also be used to reduce antibody non-specific bindings in allantibody-based methods. The reduction of antibody non-specific bindingis owing to the fact that the ACE methods can artificially, preciselyand specifically create and/or expose the ACE, while breaking up theantibody non-specific binding structures as described below in Section2, ACE antibodies, Example 4 and FIG. 13).

EXAMPLES OF THE ACE ANTIGEN DESIGN METHODS Example 1 A SUMO-To-ProteinConjugation Site-Specific ACE Structure Design

A SUMO can conjugate to many proteins including itself by an isopeptidebond. As demonstrated in FIG. 1A, a SUMO-to-SUMO conjugation site isbetween one SUMO C-terminal end glycine (G) and another SUMO internallysine K11. Based on the ACE methods, a GluC-cleaved SUMO-to-SUMOconjugation site-specific hapten can be designed asGVK(GGTQQQFVDITDC)TE, wherein “C” is added to the hapten and conjugatedto an immunogenic carrier, including, but not limited to, KLH or BSA tomake the hapten a complete antigen. The conjugation site-specificantibody to GVK(GGTQQQFVDITDC)TE can be made with the complete antigenand purified with the hapten-conjugated resin or other supportivematerials [e.g., GVK(GGTQQQFVDITDC-resin) TE]. The non-conjugation siteantibodies can be removed by non-conjugated linear peptide-linkedresin(s), e.g., (resin-GVKTE) and (GGTQQQFVDITDC-resin). After treatmentwith a protease or agent such as the designated protease (e.g., GluC) toartificially, specifically and precisely create and/or expose theGluC-cleaved ACE structure in a sample, the SUMO-to-SUMO K11 conjugationsite can then be detected with the ACE antibody by any antibody-basedmethods.

FIG. 1B shows an alternative design, by the same ACE method, ofSUMO-to-SUMO K11 conjugation site-specific ACE antigen. A SUMO-to-SUMOK11 conjugation site-specific hapten can also be designed asEGVK(GGTQQQGGGC)TENN, wherein “C” is added and conjugated to animmunogenic carrier including, but not limited to, KLH or BSA to makethe peptide hapten a complete antigen; wherein “GGG” next to “C” is aspacer between the hapten and the immunogenic carrier; whereinEGVK(GGTQQQ)TENN is a truncated form of the trypsin-cleaved conjugationsite-specific peptide“EGVK(GGTQQQFVDITDEDEMELQAPTDTENIPQGDF)TENNDHINLK”; and wherein theselection of a truncated form as a trypsin-cleaved ACE structure isconsistent with the fact that a peptide epitope generally consists of6-12 amino acids and this structure is preserved after trypsintreatment. The conjugation site-specific antibody toEGVK(GGTQQQGGGC)TENN can be made with the complete antigen and purifiedwith hapten peptide-conjugated resin [e.g., EGVK(GGTQQQGGGC-resin)TENN].The non-conjugation site-specific antibodies can be removed bynon-conjugated linear peptide-linked resin(s), e.g., (GGTQQQGGGC-resin)and (EGVKTENNC-resin). After treatment with a protease or agent such asthe designated protease (e.g., trypsin) to artificially, specificallyand precisely create and/or expose the ACE structure in a sample, theSUMO-to-SUMO K11 conjugation site can then be detected with the antibodyby any antibody-based methods.

FIG. 1C shows a design of SUMO-to-NF-kappa-B essential modifier (NEMO)conjugation site-specific ACE antigen by the ACE methods. NEMO issumoylated at an internal lysine K277 (Homo sapiens) by SUMO1. A K277conjugation site-specific hapten can be designed as CGGGALVAK(GGTQ)QE,wherein “C” is added to the peptide hapten and conjugated to KLH to makethe hapten a complete antigen; and wherein “GGG” is a spacer. The K277conjugation site-specific antibody to ALVAK(GGTQ)QE can be made with thecomplete antigen and purified with the hapten-conjugated resin [e.g.,resin-CGGGALVAK(GGTQ)QE]. The non-conjugation site-specific antibodiescan be removed by non-conjugated linear peptide-linked resin(s), e.g.,(resin-CGGGALVAKQE) and (resin-CGGTQ). After treatment with a proteaseor agent such as the designated protease (e.g., GluC) to artificially,specifically and precisely create and/or expose the GluC-cleaved ACEstructure in a sample, the SUMO-to-NEMO conjugation site can then bedetected with the antibody by all antibody-based methods.

FIG. 1D shows an ACE design of SUMO-to-heat shock factor protein 1(HSF1) conjugation site-specific antigen. HSF1 is sumoylated at a lysineresidue K298 (Homo sapiens) by SUMO2/3. A conjugation site-specifichapten can be designed as RVK(GGTQQQGGC)E, wherein “C” is added to thepeptide hapten and conjugated to an immunogenic carrier including, butnot limited to, KLH or BSA to make the hapten a complete antigen;wherein “GG” is a spacer; and wherein “RVK(GGTQQQ)E” is the ACEstructure. The K298 conjugation site-specific antibody to RVK(GGTQQQ)Ecan be made with the complete antigen and purified with thehapten-conjugated resin [e.g., RVK(GGTQQQGGGC-resin)E]. Thenon-conjugation site-specific antibodies can be removed by thenon-conjugated peptide-linked resin(s), e.g., (resin-RVKE) and(resin-CGGTQQQ). After treatment with a protease or agent such as thedesignated protease (e.g., GluC) to artificially, specifically andprecisely create and/or expose the ACE structure in a sample, theSUMO2/3-to-HSF1 K298 conjugation site can then be detected with theantibody by all antibody-based methods.

FIG. 1E shows an ACE design of SUMO-to-hypoxia-inducible factor 1 alpha(HIF1alpha) conjugation site-specific antigen. HIF1alpha is sumoylatedat a lysine residue K391 (Homo sapiens) by SUMO2/3. A K391 conjugationsite-specific peptide hapten can be designed as LK(GGTQQQGGC)K, wherein“C” is added to the hapten and conjugated to an immunogenic carrierincluding, but not limited to, KLH or BSA to make the hapten a completeantigen; and wherein “GG” is a spacer. The conjugation site-specificantibody to LK(GGTQQQ)K can be made with the complete antigen and thenpurified with hapten-conjugated resin [e.g., LK(GGTQQQGGGC-resin)K]. Thenon-conjugation site-specific antibodies can be removed bynon-conjugated linear peptide-linked resin(s), e.g., (resin-CLKK) and(resin-CGGTQQQ). After treatment with a protease or agent such as thedesignated protease (e.g., trypsin) to artificially, specifically andprecisely create and/or expose the ACE structure in a sample, theSUMO2/3-to-HIF1alpha K391 conjugation site can then be detected with theantibody by all antibody-based methods.

FIG. 1F shows a hydrolysis-guided ACE design of SUMO-to-p53 conjugationsite-specific antigen. The p53 tumor suppressor is sumoylated at alysine residue K386 (Homo sapiens) by SUMO1. A K386 conjugationsite-specific peptide hapten can be designed as CGGGKLMFK(GGTQ)TE whichis derived from the GluC-cleaved peptidePGGSRAHSSHLKSKKGQSTSRHKKLMFK(GGTQ)TE, wherein “C” is added to the haptenand conjugated to an immunogenic carrier including, but not limited to,KLH or BSA to make the hapten a complete antigen; and wherein “GGG” is aspacer. The ACE antibody to KLMFK(GGTQ)TE can be made with the completeantigen and then purified with hapten-conjugated resin [e.g.,resin-CGGGKLMFK(GGT)K]. The non-conjugation site-specific antibodies canbe removed by the non-conjugated peptide-linked resin(s), e.g.,(resin-CKLMFATE) and (resin-CGGTQ). After treatment with a protease oragent such as the designated protease (e.g., GluC) to artificially,specifically and precisely create and/or expose the ACE structure in asample, the SUMO1-to-p53 K386 conjugation site can then be detected withthe antibody by all immunological methods.

Example 2 Protein-To-Lipid (Protein Lipidation) ACE Antigen Design

A The ACE methods can be used to design protein lipidation conjugationsite-specific antigens. Exemplary protein-to-lipid conjugations include,but are not limited to, the microtubule-associated protein 1A/1B-lightchain 3 (LC3); gamma-aminobutyric-acid-type-A-receptor-associatedprotein (GABARAP); and Golgi-associated ATPase enhancer of 16 kDa(GATE16). Activation of these proteins requires lipidation of theC-terminal glycine (G) with phosphotidylethanolamine (PE). Therefore,antibodies specific to LC3-to-PE, GABARAP-to-PE, or GATE16-to-PEconjugation sites will be very useful in assays of the active forms andthe autophagy pathway activities in physiological and pathologicalconditions. Unfortunately, such antibodies are not currently available.The inventive ACE methods can be used in this situation to design anddetect the lipidated form-specific ACE epitope/antigens.

FIG. 2 shows an example of designs of LC3II-specific ACE antigen: Method1a shows that TFG-phosphotidylethanolamine (PE) with a base sequence setforth by SEQ ID NO: 14 is synthesized with a peptide synthesizer and isused to make a liposome carrier for immunization. The LC3II-specificantibody can be made and purified with TFG-PE-containing liposome, bythe method described in the book “Liposome Technology: Vol. III:Interactions of Liposomes with the Biological Milieu” By GregoryGregoriadis, CRC Press, 1993. The shaded area indicates the expected ACEstructure. Method 1b illustrates how the TFG-PE ACE structure isartificially, specifically and precisely created and/or exposed by GluCtreatment and detected by the ACE antibody in a lipid membrane samplepreparation.

Methods 2a, 2b and 2c of FIG. 2 show GluC-, CNBr- and trypsin-cleavedLC3P-PE-specific ACE antigen designs: TFGGGC; VYASQETFGC; andDEDGFLYMVYASQETFGC (SEQ ID NOS: 14-16), respectively, wherein “GGG” inthe TFGGGGC peptide is a spacer; and wherein the terminal “C” is addedfor conjugation to an immunogenic carrier. The LC3-PE-specificantibodies to base sequences set forth by SEQ ID NOS: 14-16 can be madewith the complete antigens and purified with the hapten-conjugatedresins, respectively. After treatment with the designated protease oragent (e.g., GluC, CNBr and trypsin, respectively) to artificially,specifically and precisely create and/or expose the corresponding ACEstructures in sample preparations, the LC3-PE active form can bespecifically detected with the (protease or agent) corresponding ACEantibodies by all antibody-based methods. Although cellular free pro-LC3and LC3 also contain the base sequences set forth by SEQ ID NOS: 14-16,without conjugation to PE/membrane, they are too small to be retained inmembrane samples or sample preparations and therefore can be washed outafter hydrolysis treatment.

Methods 3 and 4 of FIG. 2 show phospholipase-D (PLD) and -C (PLC)cleaved LC3-PE-specific ACE antigen designs with a base sequence setforth by SEQ ID NO: 17: CGGGSQETFG-ethanolamine (for PLD) andCGGGSQETFG-phosphoethanolamine (for PLC), wherein “C” is added forconjugation to an immunogenic carrier, and “GGG” is a spacer. LC3-PElipidation form-specific antibody can be made with the complete antigensand purified with the ACE hapten conjugated resins. After treatment withPLD or PLC to artificially, specifically and precisely create and/orexpose the ACE structures in samples, the LC3-PE conjugated forms canthen be detected with their (lipase) corresponding ACE antibodies by allantibody-based methods.

Example 3 Glycosylated Protein ACE Antigen Design

Alpha-fetoprotein (AFP) is glycosylated at the single asparagine (N)residue by a polysaccharide (N-link). Addition of a fucose to theinnermost (also referred to as core) N-acetylglucosamine (GlcNAc) of AFPglycan via the alpha(1,6) bond (i.e., core-fucosylated AFP or Fuc-GlcNAcAFP) is seen mainly in cells, body fluids and tissues of many types ofcancers, such as hepato-cellular carcinoma (HCC), thus being referred toas cancer-type AFP (Otake et al., 2001). Recent studies show thatcore-fucosylation of other proteins occurs also in pancreatic, prostate,colon, lung, and gastrointestinal cancers (Kossowska et al., 2005; Hu etal., 2008; Miyoshi et al., 2010; Osumi et al., 2009; Narisada et al.,2010; Moriwaki et al., 2010; Saldova et al., 2010; Wu et al., 2010). Forthat reason, the core-fucosylation conjugation site-specific antibodiesto cancer tissue-specific proteins are very useful in R&D, diagnosticsand therapeutics of different types of cancers. However, this type ofantibodies cannot be made with general antigens because the antigenscannot be detected owing to steric hindrance from the long and foldedpolysaccharide chain and its conjugated protein, and thus, has not beenavailable until now.

We produced the core-fucosylated AFP antibody by the ACE methods withthe following steps: The glycoform-specific ACE haptens were designed asCGGGKVN(GlcNAc)FTEI (SEQ ID NO: 1) (for the non-cancer types (non-corefucosylation types), and CGGGKVN[alpha(1,6) Fuc-GlcNAc]FTEI (SEQ IDNO: 1) for the cancer types (core-fucosylation types), respectively,wherein GlcNAc or alpha(1,6) Fuc-GlcNAc was linked to the sidechain ofthe asparagine (N) of AFP protein; and wherein CGGGKVN(GlcNAc)FTEI andCGGGKVN[alpha(1,6) Fuc-GlcNAc]FTEI were the ACE structures/segmentsspecifically cleaved by Endo-N-acetyl-beta-D-glucosaminidase-D, -H or -F(EndoD/H/F). Conjugation site-specific antibodies toCGGGKVN(GlcNAc)-FTEI or CGGGKVN[alpha(1,6) Fuc-GlcNAc]FTEI were madewith the KLH-conjugated complete antigen and purified with thehapten-conjugated resins. Non-conjugation site pan antibodies wereremoved with the non-glycopeptide-conjugated resins. After treatmentwith EndoD/H/F to artificially, specifically and precisely create and/orexpose the hidden ACE structures in a sample, the cancer type andnon-cancer type of AFP can be detected with their corresponding ACEantibodies by all antibody-based methods (see FIG. 3).

The method of detecting the core-fucosylated AFP with theglycoform-specific antibody is only an example of the invention. Otherglycoproteins with the same core-fucosylation in normal and pathologicalconditions can be found in the literature (e.g., Goldfarb et al., 1986;Sekine et al., 1987; Matsumoto et al., 1994; Naitoh et al., 1999;Yamashita et al., 1989; Bunkenborg et al., 2004; Block et al., 2005;Mehta and Block, 2008; Communale et al., 2006; Sessom et al., 2008; deLeoz et al., 2008; Li et al., 2009; Szajda et al., 2008; White et al.,2009; Cao et al., 2009). Comprehensive information about updatedcore-fucosylated glycoproteins can also be found in publicallyaccessible databases. However, conjugation site-specific antibodies tothese glycoproteins are not previously available. The inventive ACEmethods of making glycoform-specific antibodies and their use thereofcan apply for detecting any glycoprotein that is currently known or willbe identified in the future in normal or pathological conditions.

FIG. 3, Method 1 shows an example of glycoform-specific ACEantibody-based immunoassays with following steps: (i) two or moredifferent glycoforms of the same protein backbone (e.g., twoglycoproteins with the same polypeptide backbone, but with differentglycan moieties) in a sample are digested with a single or a combinationof endoglycosidases including, but not limited to, Endo D/H, which canartificially, specifically and precisely create (free terminals) and/orexpose the innermost N-acetylglucosamine (GlcNAc), the innermost corefucosylated N-acetylglucosamine (Fuc-GlcNAc), or any other modifiedinnermost GlcNAc on proteins; (ii) the digested mixture containing(monosaccharide) GlcNAc-and (disaccharide) Fuc-GlcNAc-glycoproteins isthen incubated on a surface precoated with a general antibody(antibody 1) to a non-glycan portion of the protein; and (iii) additionsof a fluorophor-1 -labeled conjugation site-specific antibody (antibody2) against the monosaccharide GlcNAc portion of the protein, and/or afluorophor-2-labeld conjugation site-specific antibody (antibody-3)against the disaccharide Fuc-GlcNAc portion of the protein. Thefluorophors can then be detected or imaged with a dual wavelengthfluorometer.

FIG. 3, Method 2 shows another example of the glycoform-specific ACEantibody immunoassays in following steps: (i) in this case, two or moredifferent glycoproteins with the same core-fucosylated N-glycan, e.g.,glycoproteins-1 and -2 in a sample, are digested with Endo D/H toartificially, specifically and precisely create and/or expose the newconjugation site-specific ACE epitope; (ii) the digested mixturecontaining disaccharide Fuc-GlcNAc-proteins-1 and-2 is incubated on asurface precoated with a antibody against allFuc-GlcNAc-asparagine-containing proteins; and (iii) additions offluorophor-1 -labeled antibody against a non-glycan portion ofglycoprotein-1, and/or fluorophor-2-labeled antibody against anon-glycan portion of glycoprotein-2. The fluorophors can then bedetected or imaged with a dual wavelength fluorometer.

FIG. 3, Method 3 shows a negative control for glycoform-specificantibody labeling. N-glycan glycoproteins are deglycosylated with PNGaseF which removes the entire N-glycans from the proteins and createsnon-glycosylated proteins in a sample. The PNGase F-deglycosylatedproteins can still bind to a general antibody against non-glycanportions of the proteins, but cannot be recognized by conjugationsite-specific antibodies to either the (monosaccharide) GlcNAc or(disaccharide) Fuc-GlcNAc portion of the proteins. This method can beused as a negative control for glycoform-specific antibody labeling.

FIG. 3, Method 4 further shows an example of Western blotting with AFPglycoform-specific antibodies. Samples were prepared fromhepato-cellular carcinoma cell culture media, treated with non-enzymesolution as a control (Ctr), a mixture of endo-D/H, or with generalPNGase-F (F), and then subjected to immunoblot analysis. Immunoblotswere labeled with: (a) a general AFP antibody to a non-glycan portion ofAFP, (b) a core-fucosylation site-specific AFP antibody, and (c) amonosaccharide GlcNAc (no core-fucosylation) AFP antibody. The generalAFP antibody detects all glycosylated and de-glycosylated AFP bands(FIG. 3, Method 4a), the core-fucosylated site-specific AFP antibodylabels only the core-fucosylated AFP (FIG. 3, Method 4b), and the(monosaccharide) GlcNAc site-specific AFP antibody labels only themonosaccharide GlcNAc AFP (FIG. 3, Method 4c).

Example 4 The Ubiquitin-To-Protein Conjugation Site-Specific Hidden ACEAntigen Design

As shown in FIG. 4, ubiquitin-to-ubiquitin conjugation site is betweenone ubiquitin C-terminal end glycine (G) and the internal (K48) residueof another ubiquitin, wherein a conjugation site-specific peptide haptencan be designed as CGGGLIFAGK(GG)QLEDGR, wherein the ubiquitinC-terminal dipeptide glycine-glycine (GG) is linked to the internallysine K48 sidechain epsilon amino group by an isopeptide bond; andwherein “C” is added for conjugation to an immunogenic carrier, and GGGis a spacer. The conjugation site-specific antibody can be made with thecomplete antigen and purified with the hapten-conjugated resin. Thenon-conjugation site pan antibodies can be removed by the linearpeptide-linked resin(s). After treatment with the designated protease(e.g., trypsin) to artificially, specifically and precisely createand/or expose the ACE structure in a sample, the hiddenubiquitin-to-ubiquitin K48 conjugation site can then be detected withthe ACE antibody by all antibody-based methods.

FIG. 4 also shows a part of the basic rationale for a hidden ACE antigendesign. The internal K48 residue of ubiquitin 1 is conjugated by theC-terminal end glycine of ubiquitin 2. The conjugation siteLIFAGK₄₈(GG)QLEDGR is hidden on the inside of the ubiquitin-to-ubiquitinconjugate structure (FIG. 4, A and B). Also, this conjugation siteLIFAGK₄₈(GG)QLEDGR region in the intact conjugation form is poorlyantigenic. Therefore, artificial and specific, rather than random oraccidental, exposure of the hidden ACE structure with a specificprotease(s) or hydrolytic agent(s) can improve the antibodyaccessibility to the antigen in a sample. In addition, the specifichydrolysis treatment can expose two free N-termini and one C-terminus.These newly created termini are charged and thus more antigenic. Ourdata show that these ACE methods are highly feasible, effective, andreliable in detecting hidden antigens in sample preparations (see FIGS.3 and 9-13). The ubiquitin K48 conjugation site-specific antibody madeby the ACE methods can specifically recognize both branched GG, and aportion of LIFAGKQLEDGR sequence. The invention also includes thenegative absorption methods to remove non-conjugation site-specificantibody.

Similarly, ubiquitin-to-ubiquitin conjugation also occurs between oneubiquitin C-terminal G (glycine) and another ubiquitin either at theinternal K6, K11, K27, K29, K33 or K63 residue, wherein conjugationsite-specific ACE haptens can be designed as CGGGMQIFVK₆(GG)TLT for K6,TGK₁₁(GG)TITLEVEPSDTIEGGGC for K11, CGGGLEVEPSDTIENVK₂₇(GG)AK for K27,AK₂₉(GG)IQDKGGGC for K29, IQDK₃₃(GG)EGIPPDGGGC for K33, or CGGGTLSDYNIQK₆₃(GG)EST for K63, respectively; wherein the ubiquitin C-terminal enddipeptide GG is linked to the K6, K11, K27, K29, K33 or K63 sidechainepsilon amino group by an isopeptide bond or any other linkages; whereinC is added for conjugation to an immunogenic carrier; wherein GGG nextto C is a spacer which may sometimes be added to the peptide hapten toincrease the hapten structural flexibility and antigenicity; and whereinthe MQIFVK₆(GG)TLT for K6, TGK₁₁(GG)TITLEVEPSDTIE for K11,LEVEPSDTIENVK₂₇(GG)AK for K27, AK₂₉(GG)IQDK for K29, IQDK₃₃(GG)EGIPPDfor K33, or TLSDYNIQK₆₃(GG)EST for K63, are truncated forms of thetrypsin-cleaved specific peptides. After treatment with trypsin toartificially, specifically and precisely create and/or expose the hiddenACE structures in samples or sample preparations, theubiquitin-to-ubiquitin K6, K11, K27, K29, K33 or K63 conjugation sitescan then be detected, respectively, with their corresponding ACEantibodies by any antibody-based method.

Similarly, ubiquitin-to-protein conjugation occurs between the ubiquitinC-terminal G (glycine) and an internal lysine (K) residue of anysubstrate protein. Two examples of the ACE antigen design are shown inFIG. 5. Example 1, hypoxia-inducible factor 1 alpha (HIF1alpha) isubiquitinated at the lysine residue K532 (Homo sapiens). Based on thefact that an antigenic epitope generally requires 6-12 amino acids, theshaded area EFK(GG)ELVEK in FIG. 5 is the designated trypsin-cleavedconjugation site-specific ACE segment. A conjugation site-specific ACEhapten can be designed as CMVNEFK(GG)ELVEK, wherein “C” is added forconjugation to an immunogenic carrier. The conjugation site-specificantibody can be made with the complete antigen and purified with thehapten-conjugated resins. After treatment with trypsin to artificially,specifically and precisely create and/or expose the hidden ACE structurein a sample, the ubiquitin-to-HIF conjugation site can then be detectedwith this antibody by any antibody-based method.

Another example is the K120-ubiquitinated histone 2A (Rattus norvegicus)(FIG. 5). A conjugation site-specific ACE hapten is designed asKK(GG)TESHHKGGGC, wherein “C” is added for conjugation to an immunogeniccarrier, and “GGG” is a spacer. The conjugation site-specific antibodycan be made with the complete antigen and purified with thehapten-conjugated resin. The non-branched peptide-conjugated resin canbe used to remove non-conjugation site pan antibodies. After treatmentwith trypsin to artificially, specifically and precisely create and/orexpose the ACE segment in a sample, the ubiquitin-to-histone conjugationsite can then be detected with this ACE antibody by all antibody-basedmethods.

Example 5 The fatty Acid-To-Protein Conjugation Site-Specific ACE Design

Similar to lipidated proteins described above in Example 2, a widevariety of cellular or viral proteins are covalently conjugated with a14-carbon fatty acid myristate(s) and/or a 16-carbon fatty acidpalmitate(s), as well as other fatty acids or lipid-related molecules.These types of protein lipidation are for associating the proteins tocell membranes from the cytoplasmic sides. Examples of making fattyacid-to-protein conjugation site-specific antibodies and their usethereof are shown in FIG. 6. Caveolin-1 is palmitoylated at threecysteine residues C133, C143 and C156 (Homo sapiens). A C133palmitoylation site-specific ACE contains a base sequence set forth bySEQ ID NO: 3 and can be designed as CGGGAVVPC₁₃₃(palmitate)IK, whereinthe N-terminal C is added for conjugation to an immunogenic carrier;wherein “GGG” is a spacer; and wherein palmitate is linked covalently tothe C133. The conjugation site-specific antibody can be generated withthe complete antigen and purified with the hapten-conjugated resins. Thenon-branched peptide-conjugated resins can be used to removenon-conjugation site pan antibodies. After treatment with trypsin toartificially, specifically and precisely create and/or expose the hiddenACE segment in a sample, the caveolin-1 C133 palmitoylation conjugationsite can then be detected with the ACE antibody by any antibody-basedmethod.

Similarly, proto-oncogene tyrosine-protein kinase Src (EC 2.7.10.2)(pp60c-src) (Homo sapiens) is myristoylated at the N-terminal endglycine residue (G2) (FIG. 6). A myristoylation site-specific ACE haptencontains a base sequence set forth by SEQ ID NO: 4 and can be designedas myristate-GSNK, wherein the N-terminal G is myristoylated, and themyristate chain can be conjugated to a liposome carrier to form acomplete antigen for immunization. The conjugation site-specificantibody can be made with the complete ACE antigen and purified withmyristate-GSNK-containing liposomes by the method described in the book“Liposome Technology: Vol. III: Interactions of Liposomes with theBiological Milieu” Gregory Gregoriadis (Editor), CRC Press, 1993. Thenon-conjugation site pan antibodies can be removed by the non-conjugatedpeptide-linked resin(s). After treatment with the designated protease oragent (e.g., trypsin) to artificially, specifically and precisely createand/or expose the hidden ACE structure in a sample or samplepreparation, the pp60c-src C133 myristoylation site can then be detectedwith the ACE antibody by any antibody-based method.

Also, FIG. 6 illustrates: (i) sonic hedgehog protein (SHH) (Homosapiens, BAA24866) that is covalently conjugated to cholesterol at theglycine residue (G197); (ii) the GTPase HRas (p2lras) (Homo sapiens)fernesylation at the 4^(th) cysteine residue (C186) from the C-terminalend; and (iii) ras-related C3 botulinum toxin substrate 1 (p21-Rac1)(Homo sapiens) geranylgeranylation at the 3rd cysteine (C 189) from theC-terminal end. Conjugation site-specific ACE antigens contain basesequences set forth by SEQ ID NOS: 5-7 and can be designed asCGGGAG₁₉₇(cholesterol)GCF for SHH; KC₁₈₆(farnesol)VLSGGGC for p2lras;and KC₁₈₉(geranylgeranyl isoprene)LLLGGGC for p21-Rac1, wherein thecholesterol, farnesol and geranylgeranyl isoprene are linked to theG197, C186 and C189 of their corresponding proteins, respectively;wherein the terminal “C” is added to the hapten for conjugation to animmunogenic carrier; and “GGG” next to “C” is a spacer. The conjugationsite-specific antibodies can be made with their corresponding completeantigens and purified with hapten-conjugated resins. The non-conjugationsite pan antibody can be removed by the non-branched peptide-linkedresin(s). After treatment with pepsin for SHH, or LysN for both p21-rasand p21-Rac1 to artificially, specifically and precisely create and/orexpose the ACE structures in sample preparations, the SHH, p21ras, andp21-Rac 1 fatty acid conjugation sites can then be detected with theircorresponding ACE antibodies by any antibody-based method.

Example 6 GPI-to-Protein Conjugation Site-Specific ACE Design

GPI is post-translationally conjugated to a group of structurally andfunctionally diversified proteins via an ethanolamine linker. This groupof proteins is anchored in the outer leaflet of the cell membrane. FIG.7 shows two examples of the ACE antigen design for making conjugationsite-specific antibodies to GPI-anchored proteins and their use thereof.One is the prion protein (PrP) (Homo sapiens) that is conjugated by aGPI-anchor at the cysteine residue C230. A conjugation site-specific ACEhapten contains a base sequence set forth by SEQ ID NO: 8 and can bedesigned as GC-phosphoethanolamine-SGGGC, wherein “SGGG” is added as aspacer and a linker that, via the serine hydroxyl side chain, links tophosphoethanolamine; wherein C is added for conjugation to KLH or BSAfor making the hapten a complete antigen. The conjugation site-specificantibody to GC-phosphoethanolamine can be made with the complete antigenand purified with the hapten-conjugated resin. After treatment withtrypsin to artificially, specifically and precisely create and/or exposethe ACE structure in a sample, the PrP-to-GPI conjugation site can thenbe detected with the ACE antibody by any antibody-based method.

Another example is the glypican-1 that is also conjugated to GPI at aserine residue S530. A conjugation site-specific ACE hapten contains abase sequence set forth by SEQ ID NO: 9 and can be designed asTS-phosphoethanolamine-SGGGC, wherein “SGGG” is added as a spacer andlinker that, via the serine hydroxyl side chain, links tophosphoethanolamine; wherein C is added for conjugation to KLH or BSAfor making the hapten a complete antigen. The conjugation site-specificantibody to GC-phosphoethanolamine can be made with the complete antigenand purified with the hapten-conjugated resin. After treatment withtrypsin to artificially, specifically and precisely create and/or exposethe ACE structure in a sample, the glypicanl -to-GPI conjugation sitecan then be detected with the ACE antibody by any immunological andantibody-based method.

Example 7 Transglutaminase-Mediated Conjugation Site-Specific ACEAntigen Design

A Transglutaminases (TGs) are a family of structurally and functionallyrelated enzymes that catalyze protein-to-protein conjugation via oneprotein's asparagine carboxamine to a lysine (K) of another protein.FIG. 8 shows an example of conjugation site-specific ACE hapten designfor making antibodies to detect transglutaminase-mediatedprotein-to-protein conjugates and their use thereofTransglutaminase-mediated synuclein-to-synuclein conjugation occursbetween the glutamine 99 (Q99) and the lysine 80 (K80) residues via ane-(g-glutamyl)lysine bridge. A trypsin-cleaved synuclein-to-synucleinconjugation site-specific ACE hapten has base sequences set forth by SEQID NOS: 10-11 and can be designed as CGGGAQK(DQLGK)TV, whereinAQK(DQLGK)TV is a truncated form of the trypsin-cleaved ACE structure;and wherein “C” is added for conjugation to an immunogenic carrier, and“GGG” is a spacer. The conjugation site-specific antibody can be madewith the complete antigen and purified with the hapten-conjugatedresins. After treatment with the designated trypsin to artificially,specifically and precisely create and/or expose the hidden ACE structurein samples or sample preparations, the synuclein-to-synucleinconjugation site can then be detected with this ACE antibody by anyimmunological method.

Another example is the amyloid beta A4 peptide (beta/A4) that is themajor component of amyloid plaque cores in Alzheimer disease, whereinthe conjugation site is between the Q15 of one amyloid beta A4 peptideto the K28 of another amyloid beta A4 peptide by an isopeptide bond. Aconjugation site-specific hapten contains base sequences set forth bySEQ ID NOS: 12-13 and can bedesigned as GSNK(YEVHHQK)GAII-GGGC via theQ-to-K conjugation, wherein C is added and conjugated to an immunogeniccarrier and GGG is a spacer. The conjugation site-specific antibody canbe made with the complete antigen and purified with thehapten-conjugated resins. The non-conjugation site pan antibodies can beremoved by the non-branched peptide-conjugated resin(s). After treatmentwith trypsin to artificially, specifically and precisely create and/orexpose the hidden ACE structures in samples or sample preparations, theamyloid beta A4 conjugation sites can be detected with this ACE antibodyby all antibody-based methods.

Comprehensive information about normal and disease-relatedtransglutaminase-mediated protein conjugation sequences can also befound in publically accessible databases including, but not limited to,Transdab at genomics.dote.hu/wiki/index.php/Main_Page.

Example 8 ADP-Ribosylation Conjugation Site-Specific ACE Antigen Design

ADP-ribosylation is a reversible posttranslational modification byadding one or more ADP-ribose moieties to protein backbone. Bothmono-and poly-ADP-ribosylated proteins involve in cell processesincluding, but not limited to DNA repair, genomic stability, andinflammation. Mono-ADP-ribosylation is initiated byADP-ribosyltransferases, which transfer an ADP-ribose residue ontoarginine, glutamic acid, or aspartic acid residues in their substrateproteins. Poly(ADP-ribosyl)ation is carried out by poly ADP-ribosepolymerase (PARP) via transferring multiple ADP-ribosyl groups ontoproteins to form long branched chains. Poly-ADP-ribosyl groups can beremoved from proteins by poly(ADP-ribose) glycohydrolase (PARG), whichcatalyzes the hydrolytic removal of the branched poly(ADP-ribose)polymer, and produces mono-ADP-ribose-protein (Gagne et al., 2005). Manybacterial toxins including, but not limited to cholera toxin, diphtheriatoxin, heat-labile enterotoxin; exotoxin A; pertussis toxin; and thelike, are ribosyltransferases and modify human target proteins byADP-ribosylation to exert their toxic effects. Therefore, bacterialtoxin ADP-ribosylated proteins can be used as biomarkers of bacterialinfections of particular diseases.

Several attempts have been made to produce antibodies to ADP-ribosylatedresidues. However, most of ADP-ribosylated residue antibodies lackspecificity, and, more importantly, these antibodies do not recognizeany specific protein poly-ADP-ribosylation sites (Liang et al., 2010).The ACE methods of this invention can be used to make an ACE antibody toa specific protein poly-ADP-ribosylation site. For example, P2Xpurinoceptor-7 (P2XR7) is an ATP receptor acting as a ligand-gated ionchannel. Poly ADP-ribosylation of P2XR7 at the R125 residue regulatesits function (Liang et al., 2010). A PARG-hydrolyzed P2XR7 ACE haptencan be designed as CPEYPTR(ADP-ribose)R with a base sequence set forthby SEQ ID NO: 2, wherein CPEYPTR(ADP-ribose)R is a segment of P2X7ribosylation site, and can be synthesized with a peptide synthesizer andconjugated to an immunogenic carrier. ADP-ribose-to-P2XR7 conjugationsite-specific ACE antibodies can be made with the complete antigen andpurified with the hapten conjugated resins. Non-conjugation site panantibodies can be removed with the linear peptide-conjugated resins.After treatment with poly ADP-ribosyl hydrolase (PARG), the P2XR7ADP-ribosylation site can then be detected with the ACE antibody by anyantibody-based method.

2. Methods of Making Ace Antibodies

The present invention further discloses methods of using ACE antigens tomake ACE antibodies. Such antibodies can be made with ACE antigens inconjunction with all antibody making methods including but not limitedto those described in the books: Antibodies -A Laboratory Manual (1988),Cold Spring Harbor Laboratory Press, and Current Protocols in Immunology(1997), John Wiley & Sons, Inc. Exemplary antibodies may be polyclonal,monoclonal, humanized, bispecific, heteroconjugate antibodies,antibody-like binding partners, and the like.

2.1. ACE polyclonal antibodies: The ACE polyclonal antibody can usuallybe made by injecting specific ACE antigens into animals including, butnot limited to, chickens, goats, guinea pigs, hamsters, horses, mice,rats, sheep and the like. Specific ACE haptens are usually linked to animmunogenic carrier including, but not limited to, KLH, serum albumin,bovine thyroglobulin, soybean trypsin inhibitor, or the like. Adjuvantsare normally used to improve or enhance an immune response to antigens.Blood serum from these animals contains polyclonal antibodies, alsoknown as antiserum, that bind to the same ACE hapten or antigen.Antigens may be also injected into chickens for generation of polyclonalantibodies in egg yolks.

2.2. ACE monoclonal antibodies: The ACE monoclonal antibody is normallyderived from a single cell line and obtained by fusingantibody-secreting lymphocytes with a cancer cell line. A mouse,hamster, rat, rabbit or other appropriate host animal can typically beimmunized with a complete ACE antigen made by attaching an ACE hapten toan immunogenic carrier. Alternatively, the lymphocytes may be immunizedin vitro. Spleen cells immunized with ACE antigens are then fused withmyeloma cells using a fusing agent to make hybridomas. A mixture ofhybridomas is then diluted and subcloned. The clones from single parentcells are then selected. The antibodies produced from the single clones(monoclonal) are then tested for their binding affinity and specificityto the antigens by any single or combinations of antibody-based methodsincluding, but not limited to, immunoblotting, immunohistochemistry,immunocytochemistry, immunoprecipitation, flow cytometry, peptide array,ELISA or all other immunoassays, or immunoelectron microscopy. Theclones with the highest binding affinity and specificity to the ACEstructures or clones for specific applications are then selected andgrown in cultures or in the peritoneal cavity of animals to a highvolume for the production of monoclonal antibodies.

Polyclonal and monoclonal antibodies can be purified using ACEhapten-conjugated matrices or resins, or by using Protein A/G orcomplete antigen-affinity chromatography for separation of antibodiesfrom other molecules in crude antibody preparations. Negativeabsorptions may be required for separating conjugation site-specificantibodies from the non-conjugation site pan antibodies, such as byusing non-branched peptide-linked resins.

2.3. ACE recombinant antibodies: The ACE monoclonal antibodies may benatural or artificial (either partially or wholly), for example,recombinant DNA methods. Recombinant monoclonal antibody involvesmolecular cloning and expression of immunoglobulin gene segments incells, viruses or yeasts. Immunoglobulin DNA expression vectors can bemade with the DNAs from hybridoma cells immunized with ACE antigen.These vectors can then be transfected into a host cells including, butnot limited to, myeloma cells in which recombinant monoclonal antibodiesare expressed.

2.4. ACE binding partners: The ACE antibodies or binding partners mayalso be made by methods including, but not limited to, phage display,yeast display, ribosome display, bacterial display, and mRNA display.

2.5. ACE humanized antibodies: The ACE antigen can be used to makehumanized antibodies or human antibodies made by recombinant methods.One approach is to merge an animal DNA sequence that encodes the smallbinding portion of a monoclonal antibody, with a human DNA sequence thatencodes the rest of the large portion of the antibody. The hybrid DNAconstruct encoding the hybrid antibodies to ACE antigens can be readilyisolated, sequenced and expressed for antibody production.

Examples of ACE antibodies: In the following immunofluorescencemicroscopic studies, tissue sections were prepared from animals perfusedwith ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS).Tissues were then postfixed in the same fixative at 4° C. for 24 h andthen sectioned with a vibratome.

Example 1

Conjugation site-specific polyclonal antibody to ubiquitin K48conjugation site was produced with rabbits. This polyclonal antibody wasdesigned to recognize the ubiquitin-to-ubiquitin K48 conjugation siteACE structure. FIG. 9 illustrates immunohistochemistry of tissuesections double-labeled with propidium iodide (PI) in a red color andwith the ubiquitin K48 conjugation site-specific antibody in a greencolor. Tissue sections were treated without (A) or with (B) trypsin.After washing and blocking with BSA, tissue sections were incubated withthe ubiquitin-to-ubiquitin K48 conjugation site-specific (primary)antibody at 4° C. overnight. Sections were then further washed anddouble-labeled with a fluorescein isothiocyanate (FITC)-labeled donkeyto rabbit secondary antibody and PI. After washing, sections weremounted onto glass slides and examined under a microscope. The ubiquitinK48 conjugation sites show the green color, and PI-stained nucleic acidsshow the red color. In FIG. 9A, because, without trypsin treatment, theubiquitin K48 conjugation site-specific ACE structures were not createdand/or exposed and thus not labeled with the ubiquitin K48 conjugationsite-specific antibody. Therefore, no green color is shown in tissuesections. In comparison, in FIG. 9B, because the K48 conjugationsite-specific ACE structures were created and/or exposed artificiallyand specifically in tissue sections by trypsin treatment, the ubiquitinK48 conjugation sites were therefore labeled in the green color with theACE antibody.

In the following Western blot analyses, tissue homogenates were preparedwith 10 volumes of 20 mmol/L HEPES, pH 8.0, 0.25 mol/L sucrose, 1.25μg/ml pepstatin A, 10 μg/ml leupeptin, 2.5 μg/ml aprotinin, and 0.5mmol/L phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride(PMSF). Protein concentration among samples was assayed and equalizedwith the buffer.

FIG. 10 shows a Western blot application of ACE antibody. Equal amountsof protein in samples were electrophoresed on 10% SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) and then transferredto nitrocellulose membranes. The membranes were blocked with BSA and0.1% Tween 20 in PBS, and then treated either with trypsin in a HEPESbuffer pH 8.0, or with the HEPES buffer only (as no trypsin control).The membranes were then incubated overnight at 4° C. with theubiquitin-to-ubiquitin K48 site-specific ACE antibody withTris/HC1-buffered saline (TBS) and 3% BSA. The membranes were incubatedwith a horseradish-peroxidase conjugated secondary antibody, and thendeveloped using an ECL system. FIG. 10A shows that ubiquitin K48conjugation sites were labeled with the ACE antibody because the hiddenubiquitin K48 conjugation site-specific ACE structures were createdand/or exposed artificially and specifically with trypsin treatment ofWestern blot membranes. The high molecular weight smeared patterns orladder bands were due to different sizes of polyubiquitinated proteins,which is consistent with the results of Western blotting polyubiquitinsmeared patterns published in the literature. FIG. 10B shows that theubiquitin K48 conjugation site-specific ACE antibody could not labelpolyubiquitin K48 conjugation sites because, without trypsin treatment,the polyubiquitin K48 conjugation site-specific ACE structures were notcreated and/or exposed.

Example 2

Immunoblot detections of hidden ACE antigens were shown in FIG. 11.Equal protein concentrations and volumes of tissue lysates weresubjected to Western blot analysis. Before antibody labeling, theWestern blot membranes were treated either without (−) or with (+)trypsin for FIG. 11A-D, or GluC for FIG. 11E. The hidden ACE structureswere designed as CIMESFK(GG)NMVPQQALVIR for Na⁺/K⁺ ATPase or ATP1A1(FIG. 11A); CHLNK(GG)MQNHGYENPTYK for Amyloid beta precursor-likeprotein 2 or Ap1p2 (FIG. 11B); CLHEDPPQK(GG)PPR for amyloid beta proteinprecursor intracellular domain associated protein-1b or AIDA-1b (FIG.11C); and CYQLFEELGK(GG)GAFSVVR (K21) for calcium/calmodulin dependentkinase II or CaMKII (FIG. 11D). The SUMO2/3-to-protein conjugation siteACE structure was designed as CGGKPKEGVK(GGTQQQ)TE (FIG. 11E). Withouttrypsin or GluC treatment (−), the specific protein bands were hiddenand thus could poorly or not be detected with their corresponding ACEantibodies. In contrast, after trypsin (for FIG. 11A-D) or GluC (forFIG. 11E) treatment (+) to artificially, specifically and preciselycreate and/or expose the ACE structures, both conjugated (arrowheads),and non-conjugated protein bands (arrows) were detected with theircorresponding antibodies.

As mentioned above, polyubiquitinated or polysumoylated protein bandshave a character of fluffy/smearing patterns or ladder bands. The ˜72kDa AIDA-1b (see FIG. 11 C) is also known as ankyrin repeat and sterilealpha motif domain-containing protein 1B or E2A-PBX1-associated protein(EB1). The protein band(s) above at 120-130 kDa is likely theubiquitinated AIDA-lb because of its fluffy pattern. This antibody alsorecognizes AIDA-1d (˜60 kDa and ˜28 kDa), and AIDA-le (˜49 kDa), asdescribed in a previous study (Jacob et al., 2010). The antibody toCaMKII labels many fluffy proteins bands which are likely the CaMKIIconjugated by varied length of polyubiquitin, and their degradationproducts (FIG. 11D). The conjugation site-specific antibody tosumoylated proteins labeled several sumoylated protein bands, which isconsistent with previous studies (e.g., Yang et al., 2008).

Example 3

A conjugation site-specific polyclonal antibody toubiqutin-to-histone-2A K120 conjugation site was produced from rabbits.This polyclonal antibody recognizes only ubiqutin-to-histone2A K120conjugation sites, and can be used in all antibody-based applications.FIG. 12 shows microscopic images of ubiquitin-to-histone K120 siteimmunostaining Tissue sections were blocked with 3% BSA in Tris-bufferedsaline (TBS), treated without (A) or with (B) trypsin, and immunolabeledwith ubiquitin-to-histone K120 conjugation site-specific ACE antibody(green) and PI (red) by the same method as that described above inExample 1. FIG. 12A shows that the nuclei in tissue sections could onlybe labeled with PI in the red color, because without trypsin treatment,the K120 conjugation site ACE structures were not artificially createdand/or exposed. In comparison, FIG. 12B illustrates both PI-stained rednuclei and K120 conjugation-site specific antibody-labeled green ACEstructures because, with trypsin treatment, the ubiquitin-to-histoneK120 conjugation sites were artificially, specifically and preciselycreated and/or exposed.

Example 4

The inventive ACE methods can significantly reduce non-specific bindingin all antibody-based applications. A key reason is that the hydrolytictreatment can artificially, specifically and precisely create and/orexpose the ACE structures while breaking up antibody non-specificbinding macromolecules. FIG. 13A shows that ubiquitin-to-histone K120conjugation sites were labeled with the conjugation site-specific ACEantiserum because the ubiquitin-to-histone site-specific ACE structureswere exposed artificially and specifically with trypsin treatment. Thearrow indicates the monoubiquitin-to-histone K120 conjugate band. Incomparison, FIG. 13B indicates that, without trypsin treatment, theubiquitin-to-histone conjugation site-specific ACE antiserum labels manyprotein bands, which are likely antibody non-specific labeling becausemolecular sizes of these bands are inconsistent with that of themonoubiquitin-to-histone K120 conjugates. In this case, theubiquitin-to-histone K120 conjugation site-specific ACE antibody was acrude antiserum. However, this piece of information suggests that theACE methods are not only able to create or expose the ACE structures inan artificial, specific and precise manner, but also likely to break upmacromolecules that otherwise bind to antibody non-specifically.Therefore, after trypsin treatment, most non-specific bands disappeared.

The feature of reducing nonspecific binding provides a further utilityof the ACE methods. This is because chief obstacles associated withconventional antigen design and antibody production are: (i) weakantigenicity, (ii) antibody poor accessibility to antigen; and (iii)antibody non-specific binding. The inventive ACE methods can minimizeall these obstacles by: (a) creating and/or exposing more antigenicN-and/or C-terminal ACE structure; (b) increasing antibody accessibilityto the artificially and specifically created and/or exposed ACEstructures; and (c) reducing non-specific bindings by the breakup ofnon-specific binding molecules.

The feature of reducing non-specific binding in the inventive methodsmay be also useful in immunoprecipitation (IP) studies. This is becausethe high levels of added immunoglobulin bands often disturb observingimmuno-precipitated proteins/molecules of interest on Western blotmembranes. The ACE methods can preserve the ACE structures whilebreaking up of added immunoglobulins on Western blot membranes, and thuseliminate unwanted immunoglobulin bands.

3. Methods of Ace Exposure and Detection

The inventive ACE methods can solve the issues inherited in thoseepitopes that are: (i) hidden/concealed within molecule(s)/structure(s)and thus poorly or not accessible to large proteins/molecules likeantibodies; (ii) poorly antigenic; and (iii) interfered withnon-specific bindings. Such epitopes include, but are not limited to,those that are folded within their parent proteins/molecules,macromolecule-to-macromolecule covalent conjugation sites,molecule-to-molecule non-covalent binding sites, proteins/molecules thatare inserted into cellular membranes, structures or organelles, andproteins/molecules that are interfered with non-specific bindings. Inaddition, the ACE structures with free terminals are more charged, andthus more antigenic than the internal sequence (Clark et al., 1969).Therefore, the ACE methods are effective in detecting hidden epitopes asshown in FIGS. 1-13.

In one embodiment, the invention provides methods of artificially,specifically and precisely creating and/or exposing ACE structures fordetection; thus improving the ACE antigenicity and antibodyaccessibility in any types of sample preparations, wherein the ACEstructures in samples or sample preparations are naturally absent orhidden, and poorly accessible to antibodies, and thus, must beartificially and precisely created and/or exposed either in samplepreparations by specifically selected hydrolytic enzymes or agents;wherein the artificially creating and/or exposing the ACE structures canbe carried out in any type of sample preparations including, but notlimited to, in vivo or in vitro, in whole or part of biological bodiesor organisms, in isolated organs or organelles, in tissues or tissuesections (with or without fixation), in isolated or cultured cells, inbody fluids or cell culture media, in tissue or cell lysates, incellular or subcellular fractions, on Western blot membranes, inchromatographic or centrifuge fractions, in biochemical assay mixtures,and the like.

The method of creating and exposing the ACE structure in a samplepreparation further comprises treating the sample preparation with afixative before treating the sample preparation with the hydrolyticenzyme or hydrolytic agent. The fixative is selected from the groupconsisting of an aldehyde, an alcohol, acetone, and osmium tetroxide,including, but not limited to, formaldehyde, paraformaldehyde, andglutaraldehyde.

The said hydrolytic enzymes and agents for artificial ACE creationand/or exposure are specific and precise, rather than random oraccidental (also see below). The enzymes and agents should be mostly thesame, but can also be very occasionally different, with the one(s) usedfor the ACE antigen design. If the different hydrolytic enzymes oragents are selected, they must preserve the ACE structures fordetection. The availability, property and chemical bond specificity ofhydrolytic enzymes and agents for ACE exposures and detections can befound in public disclosures, publications/literatures, and websitesincluding, but not limited to: www.expasy.ch/tools/peptidecutter.

Other methods to improve the accessibility of a hidden epitope are touse detergents (such as Triton X100 or SDS), different pH solutions, orphysical measures such as heat to treat sample before performingantibody-based detections. These methods are principally and profoundlydifferent, and usually less, if any, effective, relative to the ACEdetecting methods. One explanation is that detergent and heat treatmentsare non-specific, random or accidental, usually cannot break covalentchemical bonds near the conjugation sites or folded molecules, andsometimes destroy (rather than expose) the epitopes. In comparison, theACE methods employ the ACE antigen to make antibodies and then use ACEantigen design information to specifically, rather than randomly oraccidentally, select residue chemical bond-specific hydrolyticenzyme(s)/agent(s) for artificially, specifically and precisely breakingthe designated chemical bonds to create (new terminals) and/or exposethe ACE structures for antibody detection. Therefore, the ACE methodsnot only fully preserve and expose the antigen structure, but alsoenhance antigenicity of the antigen structure by creating two (for alinear hidden antigen) or three (for a branched conjugation site)antigenic/charged terminals.

A number of antigen retrieval (AR) protocols have been published (Shi,2011). To date, these protocols were solely for immunochemistry (ICC) orimmunohistochemistry (IHC), and have been applied predominantly toarchival “paraffin blocks” for IHC in diagnostic surgical pathology(Shi, 2011). Many antibody reagent companies also have antigen retrievalprotocols on their websites. However, these protocols are not based onthe ACE antigen design and detection described in this application,rather, they are based on random AR attempts and/or reversal of proteinformaldehyde adducts and cross-links formed in the course of tissuefixation (Kuhlmann and Krischan, 1981). Therefore, these protocols areused solely for IHC or ICC and usually with a very low success rate, andhave potential to destroy the antigen of interest. Therefore, theeffectiveness of these protocols is accidental and unpredictable.

For example, the Abcam's protocol acknowledges that “most formalin-fixedtissue requires an antigen retrieval step before immunohistochemicalstaining can proceed. This is due to the formation of methylene bridgesduring fixation, which cross-link proteins and therefore mask antigenicsites.” The Ihcworld's protocol (www.ihcworld.com) describes that “theuse of enzyme digestion method may destroy some epitopes and tissuemorphology”. The protocol of R&D (www.rndsystems.com) recognizes that“the disadvantages of enzyme digestion method are the low success ratefor restoring immunoreactivity and the potential for destroying bothtissue morphology and the antigen of interest.” It is also noted in theMillipore's protocol (www.millipore.com) that “the listed (enzymedigestion) procedure is only suggested; no warranty or guarantee ofperformance of the above procedure is made or implied”.

For pathologists and morphologists, “seeing is believing” and mostcancers are diagnosed by morphologic methods. O'Leary et al. (2010) andShi (2011), two pioneers in the AR research, have suggested: “the ARtechnique is in many ways still in the developing stage. Furtherdevelopment of the AR technique must be based on a better scientificunderstanding of the molecular mechanisms, which represents the keypathways to improved cell/tissue sample preparation and standardizationof IHC in clinical diagnostic applications.”

The inventive ACE methods can robustly improve immunolabeling not onlyfor IHC or ICC, but all antibody-based preparations including, but notlimited to, in tissues or tissue lysates, cellular or subcellularfractions, Western blot membranes, chromatographic or centrifugefractions, and the like (see FIGS. 3 and 9-14).

An additional step of the ACE methods requires artificially,specifically and precisely create (terminals) and/or exposure of the ACEstructure before detection. At first glance, this seems an additionalstep in compared with conventional antibody detection methods. However,in practice, this step can breakup non-specific binding molecules, thusreducing non-specific bindings significantly in all antibody-basedapplications (see FIG. 13).

Another issue may be that artificial creation of new terminals and/orexposure of the ACE structure may change the size of theprotein/molecule to be detected. This obstacle can be overcome byseparation of samples first by, for instance, Western blotting, followedby ACE structure exposure (in situ) on Western blot membranes withspecifically selected hydrolytic enzyme(s) or agent(s) for detection(see FIGS. 9-13). For immunohistochemistry, ACE in tissue sections canbe exposed directly in situ with the specifically selected hydrolyticenzyme(s) or agent(s). After washing, the artificially exposed ACEstructures can then be detected by the ACE antibody (see FIGS. 9-13).For immunoassays, regular two-antibody sandwich methods can be used,i.e., a general antibody binds the non-conjugation sites or the outsideof the ACE structure, whereas the ACE antibody binds the conjugationsites or the linear ACE structures.

A host of residue chemical bond-specific hydrolytic enzymes or chemicalagents including, but not limited to, proteases, glycosidases,lipases/phospholipases, poly(ADP-ribose) hydrolases, nucleases, and thelikes, are available and can be specifically selected for the ACEmethods. The criteria for selecting specific hydrolytic enzymes oragents for the ACE antigen design/detection depend on the molecularsequence of the ACE structure, and the substrate chemical bond-cleavingspecificities, and the ACE organization, size and antigenicity. Theselecting criteria also depend on which antibody-basedmethods/applications will be used. For Western blot analysis, forexample, it is not ideal to select an enzyme that creates too smallpieces of ACE structures. Therefore, the use of the ACE methods requiresunderstanding the structure, organization and location information ofhidden antigens, the properties of hydrolytic enzymes or chemicalagents, and which antibody-based methods/applications are used.

A limitation of the inventive ACE methods in immunoprecipitation (IP)studies may be that the ACE antibody may immunoprecipitate thehydrolytic fragments, rather than entire macromolecule-to-macromoleculeconjugates or linear hidden antigens. This limitation can be overcome byIP of molecular conjugates with non-conjugation site-specificantibodies, followed by separation of the IP protein(s), for instance,by Western blotting. The ACE structures of the IP protein(s)/molecule(s)can then be artificially and specifically created and/or exposed by thechemical bond-specific hydrolysis, and be specifically detected with theACE antibodies. Another potential limitation of the ACE detectionmethods may be that ACE antigen is sometime too small to be detected bytwo antibodies in the classical two antibody sandwich enzyme-linkedimmunosorbent assay (ELISA) methods due to stereo hindrance effect. Thispotential limitation can also be overcome by selecting different ACEstructures (see ACE design), by using a competitive immunoassay or ELISAmethod, or by ACE elution/extraction methods, followed by ACE detectionincluding, but not limited to: (a) conventional IP method followed byACE elution; (b) the surface liquid ACE extraction methods described inthe website www.advion.com and by Kertesz et al. (2010); (c)antibody-based nanoparticle ACE extraction methods (Gupta et al., 2007;Chan et al., 2008); and the likes. After extraction, ACE can be detectedby any methods including, but not limited to, all types ofchromatography, spectroscopy, and mass spectrometry.

4. Utilities of Ace Mehtods, Reagents, Antibodies, Immunoassays and Kits

In the era of “-omics”, including but not limited to genomics,proteomics, glycomics, metabolomics and histomics, our knowledge ofstructures, sequences, molecular conjugation and modifications, as wellas properties of hydrolytic enzymes and chemical agents has grownexponentially. This provides an exceptional opportunity for using theinventive ACE methods, antibodies, reagents, immunoassays and kits todetect, in a conjugation site-specific manner, posttranslationalmodifications including but not limited to glycosylation, lipidation,ubiquitination, sumoylation, other UBLs, as well as any types ofpost-translational modifications of numerous individual macromolecules.

The present invention encompasses various utilities and applications ofthe ACE methods including, but not limited to: (i) research anddiscovery (R&D), (ii) diagnosing diseases, monitoring of disease stageand response to treatment, and disease prognosis, (iii) screening oftherapeutic agents, (iv) determining conjugation or de-conjugationenzyme and agent activities, (v) detecting hidden antigens that arenormally difficult to be detected by general antibody-based methods,(vi) reducing antibody non-specific bindings in all antibody-basedmethods, (vii) therapeutic applications for treatment of abnormalmolecular conjugation or de-conjugation-related diseases, and (viii)bio-materials.

4.1. Diagnostic Applications of the ACE Methods, Antibodies, Reagents,Immunoassays and Kits

Abnormal macromolecular conjugations occur in many diseases, and can beused as disease-specific biomarkers. However, antibodies tomacromolecule-to-macromolecule conjugation sites are difficult to makeby the conventional antibody design and detecting methods, because, asdescribed above, most, if not all, macromolecule-to-macromoleculeconjugation sites are hidden antigens, and thus they are not currentlyavailable. The inventive methods of designing and detecting hidden ACEantigens can therefore be used for disease diagnosis, staging,monitoring progress and treatment, and prognosis. The following are afew examples:

4.1.1. Diagnosis of lysosomal storage diseases: Lysosomal storagediseases (LSDs) are a group of approximately 40 rare inherited metabolicdisorders that result from defects in lysosomal function, The incidenceof LSDs, together as a group, is about 1:5,000-1:10,000. LSDs includingbut not limited to Danon disease, Pompe disease, X-linked myopathy andbowel diseases occur mostly as a result of deficiency of a single enzymerequired for the metabolism of lipids and glycoproteins.

The common diagnostic features include: (a) accumulation of the lipidsand glycoproteins in the cell or body fluids; and (b) accumulation ofautophagosome biomarker protein LC3II.

The conjugation site-specific ACE antibodies of the invention can beused for diagnosis of LDS by determining the autophagosome biomarkerLC3II, and specific glycoproteins or lipidated protein in tissues orbody fluids.

4.1.2. Diagnosis of neurological, neurodegenerative and conformationaldiseases: The presence of positive ubiquitinated aggregates is a commonhallmark of neurological and neurodegenerative diseases including, butnot limited to, Alzheimer's disease (AD), Parkinson's disease (PD),Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), priondiseases, as well as vascular diseases, diabetes mellitus, and the like.For instance, stroke induces significant increase in ubiquitinatedproteins in brain tissue, blood and cerebrospinal fluid (CSF). ADaggregates contain ubiquitin-conjugated beta-amyloid and Tau proteins(Cripps et al., 2008) and AD CSF has “paired helical filaments antigen”(Kudo, 1998; Mehta et al., 1985; Perry et al., 1989). PD aggregatesconsist of ubiquitin-conjugated alpha-synuclein and/or parkins HD hasubiquitin-conjugated polyglutamine repeat aggregates. ALS hasubiquitin-conjugated SOD-containing aggregates and TAR DNA bindingprotein (TDP-43). Prion diseases have ubiquitin-conjugated prionaggregates in the tissue, CSF and sera, but each disease has its ownspecific ubiquitin-conjugated proteins as indicated above.

Unfortunately, the ubiquitin-to-protein conjugation site-specificbiomarkers described above can only be detected by a combination of manysophisticated methods that are not applicable to regular clinicallaboratory settings. As a result, no reliable laboratorydisease-specific biomarkers are currently available for diagnosingneurodegenerative diseases in living patients, which is critical forstopping disease progression. Early AD and other neurodegenerativedisease symptoms are subtle, and thus the clinical diagnosis mostlyrelies on postmortem histopathology examination, from which only aportion of patients can be diagnosed. For that reason, developing earlystage disease-specific diagnostic biomarkers that can unambiguouslydiscriminate among different neurodegenerative diseases is a key stepfor developing early therapeutic interventions of cell degeneration.

Although still in the infancy, some proteins have been identified in CSFand circulating blood as potential biomarkers of neurodegenerativediseases. For example, high levels of “paired helical filaments antigen”in AD patient CSF can be recognized by antibody against residues 64-76of ubiquitin, suggesting that CSF of AD patients contains polyubiquitinconjugates (Kudo, 1998; Mehta et al., 1985; Perry et al., 1989).

TAR DNA-binding protein 43 (TDP-43) regulates transcription and splicingand is a major component of ubiquitin-positive and tau-negative neuronaland glial inclusions in the most common form of frontotemporal lobardegeneration (FTLD) and in ALS. High level of ubiquitin-conjugatedTDP-43 can be detected in CSF from FTLD and ALS patients. It has beendemonstrated that different TDP-43 profiles correspond to clinicalphenotypes of FTLD or ALS subgroups. The differential diagnosticpotential of TDP-43 was therefore suggested (Jellinger, 2009; Steinackeret al., 2008; Kasai et al. 2009; Bian and Grossman, 2007).

In addition to CSF, general ubiquitin positive immunoreactivities havebeen detected in nervous and non-nervous tissues virtually in allneurodegenerative disorders, or all other protein conformationaldiseases (Dickson, 2005). These include, but are not limited to,ubiquitin immunopositive amyloid plaques and neurofibrillary tangles inAD, alpha-synuclein-containing aggregates and Lewy bodies in PD,ubiquitin-TDP43 aggregates in ALS and FTLD, Huntingtin nuclear inclusionin HD, and ubiquitin immunoreactivity in and around areas withspongiform changes in prion diseases. The types and manifestations ofubiquitin positive immunoreactivities in neurodegenerative diseases arequite complicated and thus hard to discriminate one disease fromanother. Therefore, new diagnostic tools that can discriminate differenttypes of ubiquitinated proteins as disease biomarkers are desperatelyneeded.

Because of the diversity of ubiquitin-conjugated proteins in tissue,cell and body fluids, conjugation site-specific antibody that recognizesa specific ubiquitin-conjugated protein and its conjugation site(s) isextremely useful for diagnosis or differential diagnosis of a particulardisease, as suggested by Iwatsubo et al. (1996) and Dickson (2005).However, such conjugation site-specific antibodies are not currentlyavailable because of no effective methods for making them againstconjugation site-specific hidden epitopes/antigens. The presentinvention provides the ACE methods and conjugation site-specificantibodies for designing and detecting disease-specificubiquitin-to-protein conjugation sites in tissue samples or body fluids,thus offering unique tools for diagnosis of specific diseases.

For example, monoubiquitination of alpha-synuclein via the E3ubiquitin-ligase SIAH leads to formation of Lewy body. Monoubiquitinatedalpha-synuclein also works as a seed to recruit PD-related proteins,including, but not limited to, ubiquitinated synphilin-1 and ubiquitincarboxyl-terminal esterase L1 (UCH-L1) (Engelender, 2008; Szargel etal., 2008). The inventive conjugation site-specific antibodies to theACE structures GLSK(GG)AK and AK(GG)EGVVAAAEK of monoubiquitinatedalpha-synuclein, and/or EGVVAAAEK(GG)-TK of ubiquitinated synphilin-1,and ubiquitinated UCH-L1 in circulating blood samples, CSF, or otherbody fluids, have great potential as a cohort of biomarkers to diagnosePD of the alpha-synuclein type. Therefore, to detect a cohort of PDbiomarkers of the alpha-synuclein type in tissue, circulating blood,and/or other body fluids, the following conjugation site-specific ACEantibodies can be used: (i) conjugation site-specific antibodies to twodifferent ubiquitin-to-alpha-synuclein conjugation sites made withGLSK(GG)AK and AK(GG)EGVVAAAEK, respectively; (ii) conjugationsite-specific antibody to ubiquitinated synphilin-1 made withEGVVAAAEK(GG)TK; (iii) conjugation site-specific antibodies to fourdifferent sites of ubiquitinated UCH-L1 (ubiquitin carboxyl-terminalesterase L1) made with MQLK₄(GG)PMEINPEMLNK, K₆₅(GG)QIEEL,QIEELK₇₁(GG)GQEVSPK, and VDDK₁₅₇(GG)VNFHFILFNNVDGHLYELDGR, respectively(Meray and Lansbury, 2007; Engelender, 2008; Szargel et al., 2008).

To detect a cohort of AD biomarkers in tissue, circulating blood, and/orother body fluids, the following conjugation site-specific ACEantibodies can potentially be used: (i) conjugation site-specificantibody to ubiquitin-to-amyloid precursor protein (APP) made with anACE peptide HLSK(GG)MQQNGYENPC; (ii) conjugation site-specific antibodyto ubiquitin-to-Tau made with an ACE peptide CHVPGGGSVQIVYK₃₁₁(GG)PVDLSK; (iii) conjugation site-specific antibody toubiquitin-to-enolasel made with an ACE peptide CIEEELGSK(GG)AK; (iv)conjugation site-specific antibody to ubiquitin-to-ubiquitin K48conjugation made with an ACE peptide LIFAGK(GG)QLEDGRC; (v) conjugationsite-specific antibody to the Toll-interacting protein made with an ACEpeptide LAK(GG)NYGMTRC; and (vi) conjugation site-specific antibody toubiquitin-conjugating enzyme E2 UbcH-ben made with an ACE peptideIDILK(GG)DKWSPALQIRC. These potential AD-specific biomarker candidatesare selected based on literature and our recent studies showing thattheir levels in brain tissues were dramatically increased in ADtransgenic animals.

A further example is to detect a cohort of HD biomarkers in tissue,circulating blood and/or body fluids. The following conjugationsite-specific ACE antibodies may be used: (i) antibody to ubiquitinatedhuntingtin made with an ACE peptide MATLEK(GG)LMK; and (ii) antibody tosumoylated huntingtin made with an ACE peptide KLMK(GGTQ)AFE (Finkbeineret al., 2008, Waelter et al., 2001, Steffan et al., 2004).

A additional example is that the conjugation site-specific antibody madewith the ACE epitope CMATLEK₆(GG)LMK (of TDP-43) may be used to detect abiomarker of frontotemporal lobar degeneration (FTLD) and ALS in tissue,circulating blood, and/or body fluids (Jellinger, 2009; Steinacker etal., 2008; Kasai et al. 2009; Bian and Grossman, 2007).

The diagnostic biomarkers listed above are only a few examples. Theinventive ACE methods can be used to make conjugation site-specificantibodies and antibodies to any hidden antigens for all biomarkers thatare currently known and will be identified in the future.

4.1.3. Diagnosis of aberrant glycoprotein-related diseases: Aberrantglycosylation of proteins changes protein function and activities, andthus can lead to diseases or be byproducts/biomarkers of diseases(Durand et al., 2000; Tong et al., 2003; Troyer et al., 2004; Valmu etal., 2006; Saffroy et al., 2007; Arnold et al., 2008; Debruyne et al.,2008; Ressom et al., 2008; Zhao et al., 2008). For instance, thefollowing diseases contain aberrant glycoproteins: I-cell disease,congenital disorders of glycosylation, leukocyte adhesion deficiencytype II, hereditary erythroblastic multinuclearity with a positiveacidified serum test, and Wiskott-Aldrich syndrome. In addition, somedisease cells, including, but not limited to, those in alcoholism andcancers, can produce unique diseased forms of glycoproteins that can beused as biomarkers for disease diagnosis, staging, monitoring treatment,and prognosis.

A classic example is aberrant glycosylation of alpha-fetoprotein (AFP)in cancer cells. AFP is an oncofetal serum protein consisting of 591amino acids (Homo sapiens) and containing only a singleasparagine-linked (N-link) polymeric carbohydrate chain (Debruyne etal., 2008). AFP is a major fetal plasma glycoprotein produced in normalembryonic tissues, but its level becomes barely detectable after birth.In hepatocellular carcinoma (HCC) and seminomatous germ-cell tumors,serum AFP is greatly increased. The reappearance of AFP in HCC patientserum is currently being used as a cancer biomarker. However, the use ofAFP as a cancer biomarker is severely limited by the fact that its levelis also increased in patients with benign liver diseases including butnot limited to hepatitis and liver cirrhosis.

There are several glycosylated forms (known as glycoforms hereafter) ofAFP. The glycoform specific for hepatocellular carcinoma (HCC) is theone with the alpha (1,6)-fucosylated innermost GlcNAc known as thecore-fucosylation, whereas the glycoform in benign liver diseases doesnot have this core alpha(1,6)-fucosylation.

The binding capacity of different AFP glycoforms varies towards lensculinaris agglutinin A (LCA) lectin. Therefore, LCA lectin has been usedto isolate three different glycoforms of AFP, namely AFP-L1 (LCAnon-reactive), AFP-L2 (LCA intermediate reactive) and AFP-L3(LCA-affinitive reactive) (Breborowicz et al., 1981). AFP-L3 contains ahigh level of the AFP glycoform with the core-fucosylation. The AFP-L3assay (owned by Wako Diagnostics, Japan) was approved as a biomarker fordiagnosis of HCC by the U.S. Food and Drug Administration in 2006.

However, the routine measurement of AFP-L3 as a biomarker is somewhathampered by relatively high cost, low sensitivity, expensiveinstrumentation, and complexity of currently available assays. Othermethods for diagnosis of HCC such as ultrasound imaging are expensiveand cannot detect the appearance of HCC with tumor masses less than 3 cmin size.

In addition to the lectin-based AFP-L3 assay, Comunale et al. (2009)published another lectin-based method for detection of the corefucosylated forms of fetuin-A and hemopexin (United States PatentApplication Number: 20070037221). The core-fucosylation of serumproteins and their lectin binding activities have been known for manyyears (Goldfarb et al., 1986; Matsumoto et al., 1994; Yamashita et al.,1989; Block et al., 2005; Communale et al., 2006; Mehta and Block,2008). The lectin-based detections of core-fucosylated serum proteins incancer patients have frequently been reported as reviewed by Hirabayashiin 2008.

Lectins are a diverse group of carbohydrate-binding proteins. Eachlectin has its own specific binding profile. Lectin-binding affinity toglycoproteins, however, is generally about 10³ lower thanantibody-binding affinity to antigen (Hirabayashi, 2008). Anotherdisadvantage of lectin-based assays is that lectin can bind to manydifferent types of glycan moieties; thus its binding specificity is lowrelative to antibodies. For example, lectin-fucose interaction is notspecific for the core fucosylation (the innermost alpha1,6-fucosylation)but also for other types of fucosylation. Furthermore, lectin-to-sugarinteraction is not protein-specific. A further practical disadvantage ofusing lectin is that, unlike antibodies, immobilization of lectinusually leads to reduction of lectin-to-glycan binding affinity andcapacity (Hirabayashi, 2008). Despite these weaknesses, lectin is theonly currently available agent to separate core-fucosylated proteinsfrom non-fucosylated ones.

Ideally, a biomarker assay should possess high specificity, affinity andcapacity, as well as, being convenient to use, high throughput, highlyreproducible and with a low cost. According to these criteria, antibodypossesses many significant advantages relative to lectin and otherbinding partners. Unfortunately, antibodies that can specificallyrecognize core-fucosylated serum proteins are currently not available.

The present invention provides unique immunoassays to detectcore-fucosylated proteins by using the core-fucosylation conjugationsite-specific ACE antibodies (see above Section 1, ACE antigen design,Example 3 and FIG. 3). For example, the glycoform conjugationsite-specific antibodies of the invention can either recognize only thecore-fucosylated peptide VN[alpha(1,6)-GlcNAc]FTEI of AFP (the HCCglycoform), or only the non-fucosylated peptide ACE VN[GlcNAc]FTEI ofAFP (the non-cancer form) (see FIG. 3). Similarly, the ACE methods canbe used to detect tissue-specific core-fucosylated proteins (see below)for diagnosis of different types of cancers. The antibody-basedimmunoassays of the present invention are specific for particularglycoproteins. and technically sound, as well as simpler, moresensitive, higher throughput. and with lower cost, relative tolectin-based assays.

As described in SECTION 1: METHODS OF DESIGNING ACE ANTIGENS, Example 3:Glycosylated protein ACE antigen design, FIG. 3 shows two examples ofglycoform-specific ACE immunoassays. Example 1: (a) two or moredifferent glycoforms of the same protein are digested with a single ormixture of endoglycosidases, e.g., Endo D/F/H, which will leave only theinnermost N-acetylglucosamine (GlcNAc) or the core-fucosylatedFuc-GlcNAc on the protein backbones; (b) the resulting (monosaccharide)GlcNAc-or (disaccharide) Fuc-GlcNAc-to-protein conjugates are incubatedon a surface precoated with a general antibody (antibody 1) to anon-glycan portion of the protein; and (c) addition of thefluorophor-l-labeled conjugation site-specific antibody (antibody 2) tothe GlcNAc portion of the protein, and/or the fluorophor-2-labeledconjugation site-specific antibody (antibody-3) unique to the Fuc-GlcNAcportion of the protein. The fluorophors are detected and quantifiedwith, for example, a dual wavelength fluorometer.

FIG. 3 example 2 further shows another type of immunoassay in which: (a)two or more different proteins with the same core-fucosylated moiety,e.g., glycoproteins-1 and -2, are digested with a single or mixture ofEndo D/F/H; (b) the resulting disaccharide Fuc-GlcNAc-proteins-1 and -2are incubated with a surface precoated with the core-fucosylationsite-specific ACE antibody generally to the common disaccharideFuc-GlcNAc-asparagine (ACE) structure; and (c) addition of afluorophor-l-labeled antibody to a non-glycan portion of glycoprotein-1and/or a fluorophor-2-labeled antibody to a non-glycan portion ofglycoprotein-2. The fluorophors can be then detected or imaged with, forexample, a dual wavelength fluorometer.

Increase in core-fucosylated proteins in cancer patient serum is notrestricted to AFP. Many other glycoproteins are also core-fucosylated inmany different types of cancers including, but not limited to, lung andpancreatic cancers, as described in previous publications (Goldfarb etal., 1986; Sekine et al., 1987; Matsumoto et al., 1994; Naitoh et al.,1999; Yamashita et al., 1989; Chang et al., 2000; Bunkenborg et al.,2004; Block et al., 2005; Communale et al., 2006; Li et al., 2007; deLeoz et al., 2008; Li et al., 2009; Szajda et al., 2008; White et al.,2009; Cao et al., 2009). For example, core-fucosylation of E-cadherin isupregulated during cancer metastasis including, but not limited to, lungcancer and colon carcinoma (Hu et al., 2008; Osumi et al., 2009).Decrease in core-fucosylated proteins in leukocyte adhesiondeficiency/congenital disorder is also observed (Sturla et al., 2005;Wang et al., 2006). Therefore, as mentioned above, specificcore-fucosylated proteins can be used as biomarkers for diagnosis ofspecific cancer types. The inventive ACE methods can be used to designand develop antibodies to detect specific core-fucosylated proteins aslisted below for diagnosis of specific types of cancers. Information oncore-fucosylated proteins in tissue, body fluids and other biologicalsamples is well documented in publically accessible databases includingbut not limited to National Center for Biotechnology Information atwww.ncbi.nlm.nih.gov/ and ExPASy at www.expasy.ch/, and in publicdisclosures, literatures and publications (e.g., Mehta and Block, 2008;Cao et al., 2009).

Some examples of core fucosylation site-specific ACE structures of theinvention include, but are not limited to, (the bold letter indicatesthe conjugated asparagine): serotransferrinNYN[Fuc(alpha1,6)-GlcNAc]KSD; alpha-1-acid glycoprotein-1:QDQCIYN[alpha(1,6)-Fuc-GlcNAc]TTYLNVQR; alpha-1-acid glycoprotein-2:QNQCFYN[alpha(1,6)-Fuc-GlcNAc]SSYLNVQR; alpha-1-antitrypsin: ADTHDEILEGLNFN[alpha(1,6)-Fuc-GlcNAc]LTEIPEAQI; alpha 2-HS glycoprotein:VCQDCPLLAPLN[alpha(1,6)-Fuc-GlcNAc]DTRVVHAAK; alpha-2-glycoprotein-1:DIVEYYNDSN[alpha(1,6)-Fuc-GlcNAc]GSHVLQGR; apolipoprotein: ADGTVNQIEGEATPVN[alpha(1,6)-Fuc-GlcNAc]LTEPAK; complement factor h (isoform 1):IPC SQPPQIEHGTIN[alpha(1,6)-Fuc-GlcNAc]SSR; haptoglobin:NLFLN[alpha(1,6)-Fuc-GlcNAc]HSEN [alpha(1,6)-Fuc-GlcNAc]ATAKDIAPT;hemopexin: SWPAVGN[alpha(1,6)-Fuc-GlcNAc]C SSALR; immunoglobulin G:GLTFQQN[alpha(1,6)-Fuc-GlcNAc]ASSMCVPDQDT; kininogen:HGIQYFNN[alpha(1,6)-Fuc-GlcNAc]NTQHSSLFMLN, orLNAENN[alpha(1,6)-Fuc-GlcNAc]ATFYFK; serotransferrin:CGLVPVLAENYN[alpha(1,6)-Fuc-GlcNAc]KSDNCEDT orQQQHLFGSN[alpha(1,6)-Fuc-GlcNAc]VTDCSGNFCL; ceruloplasmin:EHEGAIYPDN[alpha(1,6)-Fuc-GlcNAc]TTDFQR; transthyretin:VVFTAN[alpha(1,6)-Fuc-GlcNAc]DSGPR; alpha-1-microglobulin:YFYN[alpha(1,6)-Fuc-GlcNAc]GTSMACETF Q; galectin-3-binding protein:PFYLTN[alpha(1,6)-Fuc-GlcNAc]SSGVD; GP-73:AVLVNN[alpha(1,6)-Fuc-GlcNAc]ITTGER; E-calherin-1:EHVKN[alpha(1,6)-Fuc-GlcNAc]STYTA orELTHGASAN[alpha(1,6)-Fuc-GlcNAc]WTIQY (SEQ ID NOs: 1 and 18 through 39).

4.1.4. p53 ubiquitination-, sumoylation-and neddylation-relatedbiomarkers. The p53 tumor suppressor functions as a transcription factorthat is maintained at low levels in unstressed cells. In response tooncogenesis, p53 levels and transcriptional activity are significantlyaltered, in part by post-translational modification, including, but notlimited to, ubiquitination, sumoylation, neddylation, phosphorylationand acetylation. The murine double minute (mdm2) oncogene-encodedprotein Mdm2 is a RING-like E3 ubiquitin ligase and acts as a negativeregulator/inhibitor of the p53 tumor suppressor. Mdm2 is up-regulated inmany tumors, and promotes ubiquitination of p53 at multiple lysineresidues, such as K370, K372, K373, K381, K382 and K386, as well asneddylation of p53 at K370, K372, and K373 residues. p53 can also besumoylated at the K386 residue.

The present invention provides methods of detecting ubiquitinated,neddylated or sumoylated p53. For example, ubiquitinated and neddylatedp53 K370, K372 and K373 site-specific ACE antigens can be designed as(i) KLH-CGGGSSHLK₃₇₀(GG)SK, (ii) SK₃₇₂(GG)KGGGC-KLH, and (iii)SKK₃₇₃(GG)GQSTRGGGC-KLH, respectively. A sumoylated p53 K386site-specific ACE antigen can be designed as LMFK₃₈₆(GGQT)TEGPDGGGC-KLH.Ubiquitined K381, K382 and K386 conjugation site-specific p53 ACEantigens can be designed as K₃₈₁(GG)K₃₈₂(GG)GQSTSRGGGC-KLH andLMFK₃₈₆(GG)TEGPDGGGC-KLH. Therefore, these ACE antibodies toubiquitinated, neddylated and sumoylated p53 may be used to monitorprogression of tumors and cellular responses to DNA damage.

4.1.5. ACE antibodies to ubiquitinated proliferating cell nuclearantigen (PCNA). PCNA is an auxiliary protein of DNA polymerase delta,and involved in the control of eukaryotic DNA replication by increasingthe polymerase's processibility during elongation of the leading strand.In response to DNA replication or damage, this protein is ubiquitinatedat K164. A ubiquitin-to-PCNA K164 conjugation site-specific ACE antibodycan be made with an ACE structure KLH-CDAVVISCAK(GG)DGVK and canpotentially be used to detect the ubiquitinated PCNA as a cancerbiomarker by all antibody-based methods.

4.1.6. ACE antibodies to ubiquitinated tubulin: A growing number ofcancer drugs including, but not limited to, taxanes, vinca alkaloids andepothilone ixapebilone can bind to tubulins and thus alter theirubiquitination. Microtubules are composed of α- and β-tubulinheterodimer filaments in the hollow cylinder. Many cancer drugs can bindto tubulins and thus alter their assembly state and increase theirdegradation by the ubiquitin proteasome system. This type of cancerdrugs has been used in the clinic for over 2 decades. The inventiveubiquitin-to-tubulin conjugation site-specific antibodies to tubulin ACEepitopes/antigens such as KLH-CANQMVK(GG)CDPR can be used to detect theubiquitin-to-tubulin biomarkers in tissues and body fluids in allantibody-based applications for monitoring tubulin-affecting drugtreatment efficiency or efficacy.

4.1.7. GPI-anchored proteins and disease biomarkers. Severalwell-established cancer biomarkers are GPI-anchored membrane proteinsincluding, but not limited to, glypican-3, carcinoembryonic antigen(CEA), semaphorins, and urokinase receptor (uPAR). GPI-anchored proteinsare often release to body fluids, including, but not limited to, bloodand CSF. A technical challenge is the low levels of GPI-anchors in bodyfluids. The inventive methods and GPI-anchor conjugation site-specificantibodies can potentially be used to detect GPI-anchored proteins in aspecific manner (see FIG. 7).

4.2. Discovery of Molecular Conjugation Sites and Sequences

In bioreagent or R&D area, ACE methods, reagents, antibodies,immunoassays and kits can be used in all antibody-based applicationsincluding but not limited to detect, identify, isolation, locate andcharacterize macromolecular conjugation sites or hidden antigensincluding, but not limited to, protein, saccharide, lipid and nucleicacid, or any combination of the above in a sample. The ACE structurescan be artificially created and/or exposed directly either in situ or exsitu on Western blot membranes, tissue sections or any other type ofbiological sample preparations.

ACE methods, antibodies, reagents, immunoassays, and kits can be useddirectly for all antibody-based separations of conjugation site-specificand any types of hidden ACE structures including, but not limited to,peptides, saccharide, lipid, nucleic acids, or any combination of theabove, followed by identification with methods of genomics, proteomics,glycomics, histomics, metabolomics, and the likes.

An example is to separate molecules in a biological sample with2-dimensional electrophoresis gel, followed by exposing ACE structureswith designated hydrolytic enzymes or agents, and then labeling thespots on the gel/blotting membranes with the ACE antibodies. The ACEpositive spots on the gel or membrane can be cut, extracted, andidentified with any mass spectrometry (MS)-related methods.

Another example is that ACE structures/segments can be captured with theACE antibodies in a mixture or biological sample lysates, and thendetected by any analytical methods. In the MS method, ACE parentmacromolecules in a sample may need to be denatured, and then digestedwith designated hydrolytic enzymes or agents, to artificially,specifically and precisely expose the ACE structures. After isolationfrom the sample with the corresponding immobilized ACE antibodies, andthen elution from antibody, the ACE structures can then be identified byMS-related methods.

A further example is to separate glyco-, lipidated, UBL-and GPI-anchoredproteins with the general ACE antibodies to the common portion of theACE structures in a sample including, but not limited to: (i) ACEantibodies recognizing the common N-linked glycoprotein ACE structures[Fuc(alpha1,6)-GlcNAc-asparagine, or GlcNAc-asparagine asparagine] (seeFIG. 3), (ii) ACE antibodies to the common GPI structures, (iii) ACEantibodies to the common lipid portion of lipidated ACE structures (seeFIG. 6), and the like. The ACE structures separated by the generalantibody can then be identified by analytical methods including, but notlimited to, MS-methods.

An additional example is to identify ACE structures in samples by themethod of antibody array-coupled peptide surface liquid extraction. Theprocedure includes: (i) coat ACE antibodies to surfaces or matricesmostly by covalent means; (ii) treat samples with specifically selectedACE hydrolytic enzymes or agents; (iii) inhibit the hydrolytic enzymesor agents with inhibitors or any other means, or separate the hydrolyticenzymes or agents from the samples by any biochemical means; (iv)incubate ACE segment-containing samples with ACE antibody-coatedsurfaces or matrices; (v) separate bound from non-bound ACE segments onthe surfaces or matrices by washing; (vi) extract bound ACE segments byappropriate liquid including, but not limited to, low pH buffers ororganic solvents; (vii) detect ACE segments in the liquid by anyanalytical means including, but not limited to, liquid chromatography,fluorescent, ultraviolet and visible spectrometry, or any MS-relatedmethods.

4.3. Utilities in enzymatic activity assays: There are a number ofapproaches for measuring enzyme activities associated with discoveriesof therapeutic agents. For example, antibody-based immunoassays ofprotein kinase activities currently represent the largest drug targetclass screened in high throughput screening (HTS) laboratories, mostlybecause phospho-specific antibodies are widely available.

Similar to protein phosphorylation, macromolecule-to-macromoleculeconjugation also plays a central role virtually in all cellularmetabolic processes. However, unlike protein kinase activity assays,there are no macromolecular conjugation site-specific antibodiescurrently available for assaying macromolecule-to-macromoleculeconjugation enzyme activities.

The present invention provides ACE methods, reagents, antibodies,immunoassays and kits for assaying macromolecular conjugation-relatedenzyme activities, modulators, cofactor, and the regulatory chemicals.The following are a few examples:

4.3.1. Methods of assays of autophagy activities with LC3II(LC3-PE)-specific ACE antibodies. The macroautophagy (autophagyhereafter) pathway is the chief route for bulk degradation of aberrantcellular contents. A biomarker unique to autophagy is the LC3II (=LC3-PEconjugates). LC3 is a mammalian homologue of yeast autophagy-relatedgene (ATG) product ATG8. LC3/ATG8 is synthesized as a pro-LC3/ATG8, andthen cleaved by ATG4 protease to become a 16-18 kDa LC3I/ATG8I. Uponactivation of autophagy, LC3I/ATG8I is conjugated or lipidated withphosphatidylethanolamine (PE). The lipidated form is the active form andreferred to as LC3II/ATG8II. The formation of LC3II conjugates iscarried out by two consecutive ubiquitination-like enzyme systems,involving ATG7 (activating enzyme) and ATG3 (conjugating enzyme) forLC3II (or LC3-PE)/ATG8II formation.

Classical methods to measure autophagy activities are quantitative EMand degradation rate of a long-lived protein. The EM method islabor-intensive and requires dedicated personnel and expensiveequipment, whereas the method of a long-lived protein turnover rate ispoorly autophagy-specific. A new biomarker unique to autophagy is theLC3II which is formed via conjugation of LC3I with PE (lipidation, seeFIG. 2). However, there is no antibody that recognizes only the activeform LC3II. The present invention provides ACE methods to detect only(lipidated or active) LC3II, and can be used to assay autophagyactivities by all antibody-based methods (see FIG. 2).

The LC3II-specific ACE antibodies of the invention can be used to assayLC3II formation-related enzyme activities including, but not limited to,ATG3, ATG7, ATG10 and ATG4, as well as these enzyme modulators,cofactors, or LC3II-related pharmacological agents. The assay systemshould contain LC3I or its peptides, PE, adenosine triphosphate (ATP),as well as ATG3, ATG7, or ATG4, or the enzyme modulators, cofactors orLC3II-related pharmacological agents. After the reaction, enzymes ortheir modulator activities as reflected by the formation of LC3II, canbe determined with the LC3II-specific antibody.

4.3.2. Methods of assays of fatty acid-to-protein conjugation enzymesand their modulators: Fatty acid-to-protein conjugations play key rolesin the localizations and functions of proteins and thus are directlyinvolved in endocytosis, cellular signaling, and many diseases. Numerouscellular and viral proteins are known to contain covalently bound lipidgroups. Myristoylation occurs at the N-terminal glycine residue byaddition of, via an amide bond, a 14-carbon saturated acyl group.Palmitoylation takes place at a C-terminal domain cysteine residue by athioester bond. Prenylation is mediated by the formation of a thioesterbond between a C-terminal domain cysteine and isoprenoids, farnesol orgeranylgeraniol. Protein prenylation substrate proteins include, but arenot limited to H-Ras, K-Ras, Rheb (Ras homolog enriched in brain),CENP-E (centromere-associated protein E), and RhoB. The C-terminalglycine residues can be covalently attached with a cholesterol moiety byan ester bond. Cell outside surface proteins can be anchored to themembrane by linking to a GPI moiety (Ferri et al., 2005).

Many proto-oncogenes including, but not limited to, pp60src family andRas family, are post-translationally lipidated and thus, they have beendrug targets for developing cancer drugs (see FIG. 6). For example,protein farnesyl transferase (FPTase) inhibitors (FTIs), proteingeranylgeranyl transferase type I (GGPTase-I) inhibitors (GGTIs), aswell as the dual prenylation inhibitors (DPIs) inhibit lipidation ofKi-ras and are currently in clinical trials for the treatment ofcancers. HDJ2 is an FPTase-specific substrate, and Rap1A is aGGPTase-I-specific substrate, and thus have been used as biomarkers formeasurement of geranylgeranylation and farnesylation activities,respectively (Lobell et al. 2002). FTIs may also be used to inhibitfarnesylation in parasites including, but not limited to, trypanosoma(African sleeping sickness) and plasmodium falciparum (malaria) (Cardosoet al., 1983).

Statins are used to inhibit cellular cholesterol and isoprenoidbiosynthesis, resulting in a decrease in protein lipidation in vivo. Forexample, statins inhibit the dimerization of beta-secretase [BACE(beta-site amyloid precursor protein-cleaving enzyme)] by inhibiting thelipidation of BACE and its associated proteins (Parsons et al., 2007).Therefore, lipidated BACE may be used as a biomarker for evaluation ofstatin treatment efficiency and efficacy.

The present invention provides methods of designing and detectinglipidation site-specific ACE structures, and can be used to evaluateprotein lipidation status and effect of protein lipidation modulators orpharmaceutical drugs in vivo. Lipidation state-specific ACE antibodiesmay further be used to assay activities of protein lipidation enzymesand their modulators. The antibody-based assays typically contain anon-lipidated protein or peptide substrate (including, but not limitedto, HDJ2 and Rap1), a lipid donor, ATP, and a lipidation enzymeincluding, but not limited to, N-myristoyltransferases (NMTs), palmitoylacyltransferases (PATs), farnesyltransferases,geranylgeranyltransferases, or the likes. After incubation, thelipidation enzyme activity as reflected by the rate of formation of thelipidated conjugates, can be determined with the lipidation conjugationsite-specific ACE antibodies. Information about enzymes and substratesof protein lipidation can also be found at: mendel.imp.ac.at;bioinformatics.lcd-ustc.org; and expasy.org.

4.3.3. Methods of assays of protein glycosylation enzymes and theirmodulators: N-linked glycoproteins are covalentpolysaccharide-to-protein conjugation via GlcNAc-to-N chemical bond. TheN-linked amino acid consensus sequence is N-X-S/T (X=any amino acidexcept proline). N-linked glycosylation is important for protein foldingand many other functions of proteins. O-linked glycoproteins are anothercovalent saccharide-to-protein conjugation via GlcNAc-to-S/T chemicalbond. Currently there is not an O-linked amino acid consensus sequence.O-linked glycosylation involves cell-to-cell adhesion by sugar complexesof proteoglycans. Proteins that circulate in the blood are not normallyO-glycosylated, with the exception of IgAl/IgD and C1-inhibitor.

Glycosyltransferases are a group of enzymes (EC 2.4) that catalyzetransferring saccharide unit from an activated sugar phosphate (known asthe “glycosyl donor”) to an acceptor molecule. Protein glycosylationbelongs to a co-translational and posttranslational modification and isprocessed in different cellular compartments, particularly in theendoplasmic reticulum (ER) and Golgi apparatus, by glycosyltransferasesand glycosidases. Most, if not all, membrane and secretory proteins areglycosylated.

The present invention provides ACE methods and conjugation site-specificantibodies for measuring protein glycosylation-related enzymaticactivities including, but not limited to, glycosidases,glycosyltransferases, and their modulators, cofactors or pharmacologicalagents. These antibody-based assays are typically carried out in asystem that contains non-glycosylated substrates including, but notlimited to, proteins or peptides, activated glycosyl donors (e.g.UDP-glucose, UDP-galaxies, UDP-GlcNAc, UDP-GalNAc, UDP-xylose,UDP-glucuronic acid, GDP-mannose, GDP-fucose, or CMP-sialic acid), ATPregenerating systems, and glycosylation-related enzymes (either naturalor recombinant), or glycosylation-related enzyme modulators, cofactors,chemical activators or inhibitors. After the reaction, the glycosylationenzyme activity as reflected by the rate of formation of theglycosylated proteins or glycosylated peptides, can then be determinedwith the conjugation site-specific ACE antibody.

4.3.4. Methods of assays of ubiquitination and de-ubiquitination enzymeactivities and their modulators: Ubiquitin is a highly conservedregulatory 76 amino acid polypeptide found in all eukaryotic cellseither free or covalently bound to other proteins. Ubiquitination (orubiquitylation) is an enzymatic, protein post-translational modificationprocess in which the carboxylic acid terminal glycine of the activatedubiquitin forms an amide bond to the epsilon amine of the lysine in themodified protein. Protein ubiquitination is carried out consecutively byubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), andubiquitin ligase (E3) to catalyze conjugation of ubiquitin to a protein.Successive conjugation of activated ubiquitin to the K-48, or K-63lysine of the previously conjugated ubiquitin form polyubiquitin chains.Polyubiquitin via K48-linkage is generally recognized by the proteasomefor degradation, whereas K63-linked polyubiquitin and monomericubiquitination is generally thought, at least in part, to function asproteasome-independent processes including but not limited toendocytosis, and regulation of enzymatic or transcriptional activities.

The present invention provides ACE methods, antibodies, reagents,immunoassays and kits for measuring activities of proteinubiquitination-related enzymes including, but not limited to, ubiquitinligases, ubiquitin hydrolases as well as their modulators, cofactors orchemical agents. The antibody-based assays of ubiquitin ligaseactivities can typically be carried out in a system that containsubiquitin and its substrates (including ubiquitin itself), a ubiquitinconjugation enzyme fraction (s) that contains E1, E2 and E3 enzymes, ATPregenerating systems, and ubiquitin-related enzymes (either natural orrecombinant), and ubiquitin-related enzyme modulators, cofactors orchemical agents. Ubiquitination-related enzymatic activities can bemeasured as the rate of formation of the ubiquitin-to-protein/peptideconjugates with their corresponding conjugation site-specificantibodies.

For example, the human double minute (Hdm2) oncogene is an E3 ubiquitinligase for ubiquitination of p53 at a lysine residue K370 (FIG. 14). TheHdm2 activity is significantly up-regulated in many type of cancers. TheACE antibody to the ubiquitin-to-p53 K370 site can be made withKLH-CGGGSSHLK₃₇₀(GG)SK). The assay mixture consists of a biotinylatedp53 peptide substrate (biotin-CGGGSSHLKSK), Hdm2, ATP, K48R ubiquitin,E1, and E2 (UbcH5c) (FIG. 14). After incubation, a stop buffer (e.g., 40mM EDTA) containing detection mix (ULight-streptavidin, and the EuropiumCryptate-labeled ubiquitin-to-p53 conjugation site-specific antibody)will be added. The p53 E3 ligase activity will be measured with atime-resolved fluorometer at excitation of 320 nm and emission 665 nm.

This TR-FRET method can be applied for all E3 ligase activityimmunoassays with the corresponding conjugation site-specificantibodies.

Similarly, determination of ubiquitin hydrolase activity can typicallybe performed in a system that contains specificubiquitin-to-protein/peptide conjugates, and ubiquitin hydrolases(either natural or recombinant), or ubiquitin hydrolase modulators orcofactors. The ubiquitin hydrolase activity can then be measured as therate of reduction of the ubiquitin-to-protein/peptide conjugates withtheir corresponding conjugation site-specific ACE antibodies.

In parallel to ubiquitin, there is a growing family of other smallposttranslation protein modifiers that conjugate to proteins in similarenzymatic pathways as those of protein ubiquitination, commonly known aseither ubiquitin-like molecules (ULMs) or ubiquitin-like proteins(UBLs). UBLs include, but are not limited to: small ubiquitin-likemodifier (SUMO), interferon-stimulated gene-15 (ISG15, also known asubiquitin cross-reactive protein), ubiquitin-related modifier-1 (URM1),neuronal-precursor-cell-expressed developmentally downregulatedprotein-8 (NEDD8, also called Rub 1 in S. cerevisiae), human leukocyteantigen F-associated (FAT10), ATG8 or LC3, and ATG12, Fau ubiquitin-likeprotein (FUB1), MUB (membrane-anchored UBL), ubiquitin fold-modifier-1(UFM1), and ubiquitin-like protein-5 [UBL5, which is also known ashomologous to ubiquitin-1 (Hubl) in Schizosaccharomyces pombe].

UBLs share only modest primary sequence identity with ubiquitin, butthey have a similar three-dimensional structure. Most UBLs use theirC-terminal glycine to conjugate a substrate protein at a lysine (K)residue by an isopeptide bond, with exceptions of yeast ATG8 and ATG12.UBLs conjugate to substrate proteins also via E1 (activating), E2(conjugating) and E3 (ligating) enzymatic systems, similar to those forprotein ubiquitination. UBL-conjugates can also be reversed byUBL-specific hydrolases in a similar fashion as that of deubiquitinatingenzymes.

The present invention provides ACE methods, antibodies, reagents,immunoassays and kits for measuring UBL-related enzyme activitiesincluding, but not limited to, UBL ligases, UBL hydrolases and theirmodulators or cofactors. The antibody-based assays can typically becarried out in a system that contains a UBL, the substrate, the E1-, E2-and E3-like conjugation enzyme fraction (either natural or recombinant),an ATP regenerating system, and UBL-related enzyme modulators orcofactors. After the enzymatic reaction, UBL-related enzyme activity asreflected by the rate of formation of the UBL-conjugated ACE structurescan be determined with their corresponding conjugation site-specific ACEantibodies.

Similarly, the methods of assays of UBL hydrolase activity can typicallybe performed in a system that contains a UBL-conjugated substrate, a UBLhydrolase (either natural or recombinant), and UBL hydrolase modulatorsor cofactors. After the enzymatic reaction, the UBL hydrolase activityas reflected by the rate of reduction of the UBL-conjugated ACEstructures, can be determined with their corresponding ACE antibodies.

5. Therapeutic Applications of the Ace Methods, Antibodies and Reagents

The ACE antibodies of the invention may be useful, for example, intargeting the conjugation sites, for treating macromolecularconjugation-and aggregation-related diseases including, but not limitedto neurodegenerative diseases, cancer, vascular diseases, inflammatorydiseases, macular degeneration, transplant rejection, multiplesclerosis, stroke, heart diseases, diabetes, infectious diseases and allprotein conjugation-related diseases.

The present invention may be relevant to the delivery of ACE antibodiesto the target by carriers including, but not limited to, liposomes. Thismay be done by packing liposomes with conjugation-site-specificantibodies and hydrolytic enzymes including, but not limited toproteases, glycosidases/deglycosylases, lipases or phospholipases,nucleases, or cytotoxic agents such as chemotherapeutic agents, toxins,or radioactive isotopes. Review articles about immunoliposome andimmunoliposome-mediated delivery can be found in publications (e.g.,Pirollo et al., 2008; Brignole and Marimpietri et al., 2005; Bendas2001; Maruyama, 2000).

The ACE antigen design of the invention may be used for preparations ofvaccines to particular diseases including, but not limited to,neurodegenerative diseases, cancers, conformation diseases (e.g. cysticfibrosis, Celiac diseases, and lysosomal storage diseases), vasculardiseases, diabetes-related diseases, and aging-related diseases. Thevaccines may be preventive or therapeutic.

6. Bio-Material Applications of the Ace Methoids, Antibodies andReagents

Conjugation site-specific or other linear hidden antigen antibodies maybe useful in biomaterial applications, such as making collagen-likebiomaterials for tissue repair. Cell-binding sequences and enzymecrosslink sites of collagen-like biomaterials are crucial in creatingcollagen mimics that can reproduce biological activities of naturalcollagens. Transglutaminase catalyzes formation of fibronectin-like, andhyaluronic acid and glycosaminoglycans-like, as well as collagen-likebiomaterials. These biomaterials are components of the tissueextracellular matrices. Conjugation site-specific ACE antibodies of theinvention may be useful in determining covalent crosslink sites ofbiomaterials.

The ACE methods, reagents, antibodies, and immunoassays can be used tomeasure hydrolytic enzymes or chemical agent activities in industrialapplications including, but not limited to cellulases and esteraseactivities in the paper industry, hydrolytic enzyme activities in theleather, pigment removal, biodegradable plastic, or bioethanolindustries, and the like.

7. Kits

In another aspect, the present invention provides kits for detecting theACEs in biological samples. Such kits comprise ACE antibodies,hydrolytic enzymes or agents, and other items including, but not limitedto secondary antibodies, enzyme modulators, cofactors, and buffersystems.

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We claim:
 1. A method of detecting a hydrolysis-created ArtificiallyCleaved Epitope (ACE) structure in a sample, wherein the ACE structureis hidden in its intact or natural form in a polymeric molecule and ispoorly accessible to antibodies, comprising steps of: (i) designing anACE structure of formula L1-L2---Ln, wherein L1 and Ln are each terminalresidues that are artificially created via chemical bond-specificcleavage by a hydrolytic enzyme or hydrolytic agent, wherein theantibody specifically recognizes at least one of the L1 or Ln terminalresidues or residues between L1 and Ln; (ii) synthesizing the ACEstructure; (iii) making an antibody against the ACE structure; (iv)creating the ACE structure in the sample by treating the samplepreparation with the hydrolytic enzyme or hydrolytic agent therebyexposing the formerly hidden ACE. structure to specific interaction withthe antibody; and (v) detecting the ACE structure created in step (iv)with the antibody, wherein the antibody specifically binds to at leastone of the artificially created L1 or Ln terminal residues or residue(s)between L1 and Ln.
 2. The method of claim 1 wherein the antibody is apolyclonal antibody, a monoclonal antibody, a bi-specific antibody, arecombinant antibody, a humanized antibody, a single chain or domainantibody, an antibody fragment, or an antibody-like molecule.
 3. Themethod of claim 1 wherein the hydrolysis-created ACE is a linearstructure selected from the group consisting of a protein segment, alipid segment, a saccharide segment, a nucleic acid segment, aprotein-to-protein conjugation segment, a protein-to-saccharideconjugation segment, a protein-to-phosphatidylethanolamine conjugationsegment, a protein-to-ubiquitin conjugation segment, a protein-to-lipidconjugation segment, a protein-to-GPI conjugation segment, aprotein-to-UBL conjugation segment, and a protein-to-(ADP-ribose)conjugation segment.
 4. The method of claim 1 wherein the hydrolyticenzyme is selected from the group consisting of a protease, aglycosidase, a lipase, a phospholipase, a nuclease, a polyribosylhydrolase, and their combinations.
 5. The method of claim 1 wherein thehydrolytic agent(s) is selected from a substance with chemicalbond-specific hydrolysis activity.
 6. The hydrolytic agents of claim 5wherein the hydrolytic agents are BNPS-skatole(3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole), CNBr (cyanogenbromide), formic acid, hydroxylamine (NH₂OH), iodosobenzoic acid, andNTCB+Ni (2-nitro-5-thiocyanobenzoic acid).
 7. The method of claim 1wherein the sample preparation is selected from the group consisting ofa blot membrane, a tissue section, an isolated organ, an isolated cell,an isolated organelle, isolated tissue, an isolated body fluid, cellculture media, cell lysate, tissue lysate, an isolated fractionsubcelhilar fraction, a chromatographic fraction, an immunocomplex, anda centrifuge fraction.
 8. The method of claim 1 wherein the step ofsynthesizing the ACE structure further comprises treating thesynthesized ACE structure with a fixative.
 9. The method of claim 8wherein the fixative is selected from the group consisting of analdehyde, an alcohol, acetone, and osmium tetroxide.
 10. A method ofdetecting hydrolysis-preserved or hydrolysis-resistant ACE structure anda method of reducing non-specific antibody binding, comprising steps of:(i) breaking up non-specific binding structures while creating ahydrolysis-preserved ACE structure by treating a sample with ahydrolytic enzyme or hydrolytic agent; and (ii) specifically detectingthe hydrolysis-preserved or hydrolysis-resistant ACE structure createdin step (i) with the antibody.
 11. The method of claim 10 wherein thehydrolytic enzyme is selected from the group consisting of a protease. aglycosidase, a lipase, a phospholipase, a nuclease, and a polyribosylhydrolase.
 12. The method of claim 10 wherein the hydrolytic agents areBNPS-skatole (3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole),cyanogen bromide (CNBr), formic acid, hydroxylamine (NH2OH),iodosobenzoic acid, and NTCB+Ni (2-nitro-5-thiocyanobenzoic acid).